PRECISION MEDICINE AND GRAFT-INDUCED DYSKINESIA (GID): 
INVESTIGATING THE CURIOUS SIDE EFFECT OF  
DOPAMINE NEURON TRANSPLANTATION IN THE rs6265 BDNF (MET/MET) 
PARKINSONIAN BRAIN 

By 

Carlye Anne Szarowicz 

A DISSERTATION 

Submitted to 
Michigan State University 
in partial fulfillment of the requirements 
for the degree of 

Pharmacology & Toxicology – Doctor of Philosophy 

2025 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ABSTRACT 

While dopamine (DA) neuron transplantation is a promising alternative therapy to 

the current pharmacological agents (e.g., levodopa) prescribed for individuals with 

Parkinson’s disease (PD), significant heterogeneity in clinical outcomes exists. 

Specifically, the underlying mechanisms responsible for the aberrant side effect, graft-

induced dyskinesia (GID), a behavior that develops in a subpopulation of individuals 

who received primary DA neuron transplants, remains a mystery to be solved. In regard 

to this heterogeneity in cell therapy, our group previously became interested in the 

influence of certain genetic risk factors, hypothesizing that the common human single 

nucleotide polymorphism (SNP), rs6265, which is found in the gene for brain-derived 

neurotrophic factor (BDNF) and results in decreased BDNF release, is an unrecognized 

contributor to response variability in cell therapy, specifically the development of GID. 

Indeed, we previously demonstrated that homozygous rs6265 (Met/Met) parkinsonian 

rats engrafted with wild-type (WT; Val/Val) DA neurons uniquely exhibited GID compared 

to their WT counterparts. To further expand these findings, I investigated the impact of 

rs6265 in both the host and donor on DA neuron transplantation for my thesis research. 

I additionally studied whether exogenous BDNF treatment would mitigate GID behavior 

in the Met/Met parkinsonian rats engrafted with WT DA neurons. In both studies, rats 

were rendered unilaterally parkinsonian using 6-hydroxydopamine (6-OHDA), engrafted 

with intrastriatal embryonic ventral mesencephalic (eVM) neurons from E14 WT or 

Met/Met donors, and assessed for amelioration of levodopa-induced dyskinesia (LID) 

(graft function) and induction of graft-induced dyskinesia (GID) (graft dysfunction). For 

the second experiment, exogenous BDNF was administered directly above the grafted 

 
 
DA neurons through a cannula connected to a subcutaneous osmotic minipump for four 

weeks following engraftment. From these experiments, I first determined that (1) the 

homozygous rs6265 Met/Met genotype, whether present in the host or donor, elicits 

superior graft-derived functional benefit compared to WT parkinsonian hosts, and (2) 

Met/Met parkinsonian rats engrafted with WT DA neurons curiously remain the only 

host/donor combination to exhibit significant GID behavior. Moreover, I discovered that 

(3) exogenous BDNF administration is not a feasible treatment for GID as BDNF 

exacerbated GID in Met/Met parkinsonian rats engrafted with WT DA neurons, and (4) 

evidence suggests that dysregulated DA/glutamate co-release and/or excess DA 

release is associated with GID induction, a phenomenon that corresponds with clinical 

trials where individuals with GID benefited from buspirone (a drug with DA antagonist 

properties) administration. Because several clinical grafting trials for PD are now 

planned or ongoing, uncovering the underlying mechanisms responsible for GID will be 

necessary to optimize cell transplantation as a safe alternative therapeutic in PD. 

Collectively, the knowledge gained from my research offers guidance moving forward for 

the development of promising precision-medicine-based therapies that effectively treat 

the majority, not only a subset, of patients with PD.  

 
 
 
 
 
Copyright by 
CARLYE ANNE SZAROWICZ 
2025 

 
 
 
 
 
 
 
 
 
 
 
 
In loving memory of my father, Robert F. Szarowicz 

v 

 
 
 
 
 
 
 
ACKNOWLEDGEMENTS 

I would first like to thank my graduate mentor, Dr. Kathy Steece-Collier, for her 

guidance over the last five years. Her expertise is vast, and I have made considerable 

strides as a scientist because of her. I, too, am grateful for the support of my lab 

manager, Jennifer Stancati. Not only has she graciously taught me laboratory 

techniques, she has also often leant a shoulder to cry on in the midst of my personal 

struggles I endured throughout the years. I would also like to thank several other lab 

members that have come and gone throughout my time here: Molly Vander Werp, Sam 

Boezwinkle, Caleb Mathai, and Asha Savani. It has been an honor developing our 

scientistic minds alongside one another. I owe additional acknowledgement to my 

committee members, including Drs. Caryl Sortwell, Anne Dorrance, John Goudreau, 

Colleen Hegg, and Margaret Caulfield, for their time and guidance as I navigated the 

doctoral program from start to finish. Thank you also to the Translational Neuroscience 

Department and the Pharmacology and Toxicology Department at MSU for providing me 

with this opportunity to pursue my doctoral degree. 

Importantly, I’d like to thank my family for their endless love and support. It is 

because of them that I made it here today. Unfortunately, my father passed away 

halfway through my fourth year here, four months after being diagnosed with Stage 4 

stomach cancer. He was my constant and would always encourage me to lift up my 

problems to God, that He would bring me peace. He was the epitome of the perfect 

father, and there was no one else like him. Today, I reached a milestone that he will 

never see. So badly, I’d like to talk to him one last time and see how proud he would be. 

vi 

 
Dad, I am honored to be your daughter, and I will miss you every day for the rest of my 

life. But I know I will see you again. I love you.  

Just like my father, my mother has been an unwavering presence throughout my 

PhD journey, offering me encouragement every step of the way. She is the strongest 

person I know. To my sister, who has loved me unconditionally since the day she was 

born, and to my brothers, who have loved me despite my sisterly flaws—thank you all.  

And lastly, but arguably most important, my husband, Jacob Kaminski. You are 

the most precious person in my life, and I am so grateful for you. You have encouraged 

me to be the best that I can be, and I cannot fathom loving you more. You have 

supported me in following my dreams without any hesitation. You are the most selfless 

person I have ever known, and I cannot thank you enough. Soon we will be bringing a 

beautiful daughter into this world, and I cannot wait to see what an incredible father you 

will be.  

Thank you, Lord, for these blessings you have brought me.  

Colossians 3:17: “And whatever you do, whether in word or deed, do it all in the name 

of the Lord Jesus, giving thanks to God the Father through him. 

vii 

 
 
 
 
 
 
 
 
PREFACE 

Upon completion of this dissertation, manuscripts derived from Chapter 3 and 4 

are both finalized and ready for submission. Chapter 3 is intended to be submitted to 

Neurobiology of Disease, and Chapter 4 is intended to be submitted to Journal for 

Clinical Investigation. Additionally, large portions of Chapter 2 were reproduced from my 

review article that was published in July 2022 in the International Journal of Molecular 

Sciences (IJMS), PMID: 35887357 (copyright is retained by the authors).  

viii 

 
 
 
TABLE OF CONTENTS 

LIST OF TABLES ......................................................................................................... xii 

LIST OF FIGURES....................................................................................................... xiii 

LIST OF ABBREVIATIONS .......................................................................................... xv 

CHAPTER 1: INTRODUCTION TO PARKINSON’S DISEASE (PD) .............................. 1 
HISTORY ..................................................................................................................... 2 
SYMPTOM PRESENTATION AND CLINICAL DIAGNOSIS ....................................... 4 
Classic Motor Symptoms ......................................................................................... 4 
Non-Motor Symptoms .............................................................................................. 6 
Clinical Diagnosis ..................................................................................................... 7 
NEUROPATHOLOGY................................................................................................ 10 
The Basal Ganglia .................................................................................................. 10 
Nigrostriatal Degeneration and DA Depletion ......................................................... 14 
Lewy Body Pathology ............................................................................................. 16 
RISK FACTORS AND ETIOLOGY ............................................................................ 19 
Advancing Age ....................................................................................................... 19 
Genetic Risk Factors .............................................................................................. 22 
Environmental Risk Factors ................................................................................... 26 
Other Risk Factors and Comorbidities ................................................................... 29 
THERAPEUTIC STRATEGIES FOR PD ................................................................... 31 
Pharmacotherapy ................................................................................................... 31 
Advanced Therapies .............................................................................................. 41 
Experimental Disease-Modifying Therapies ........................................................... 42 
Regenerative Cell Transplantation Therapy ........................................................... 48 
BIBLIOGRAPHY ....................................................................................................... 79 

CHAPTER 2: ADVANCING CELL-BASED THERAPY FOR PARKINSON’S DISEASE 
THROUGH THE SCOPE OF PRECISION MEDICINE ............................................... 125 

UNDERSTANDING THE COMPLEXITY OF PATIENT RESPONSE TO PD 
THERAPY ............................................................................................................... 126 
Introduction to Precision Medicine ....................................................................... 126 
Precision Medicine in Parkinson’s Disease .......................................................... 127 
Heterogeneity in Clinical Response to PD-related Therapy ................................. 129 

ROLE OF BDNF IN HETEROGENETIY OF CLINICAL RESPONSE TO PD 
THERAPY ............................................................................................................... 130 
Introduction to BDNF ............................................................................................ 131 
BDNF Gene Structure and Isoform Processing .................................................... 132 
BDNF Sorting and Release .................................................................................. 136 
BDNF Signaling .................................................................................................... 137 
PD and BDNF ...................................................................................................... 142 
Utilizing BDNF as a Potential Therapeutic ........................................................... 145 
Genetic Polymorphisms of BDNF ......................................................................... 152 

ix 

 
HETEROGENEITY IN SIDE EFFECT LIABLITY OF  
CELL TRANSPLANTATION ................................................................................... 156 
GID and the rs6265 BDNF SNP ........................................................................... 156 
Goals of Current Research .................................................................................. 156 
BIBLIOGRAPHY ..................................................................................................... 159 

CHAPTER 3: PRECISION MEDICINE IN PARKINSON’S DISEASE: HOST/DONOR 
INTERACTIONS AND GRAFT-INDUCED DYSKINESIA LIABILITY IN HOMOYZGOUS 
rs6265 (MET/MET) BDNF PARKINSONIAN RATS .................................................... 181 
ABSTRACT ............................................................................................................. 182 
INTRODUCTION ..................................................................................................... 183 
METHODS ............................................................................................................... 187 
Animals ................................................................................................................ 187 
Experimental Design and Timeline ....................................................................... 188 
Nigrostriatal 6-OHDA Stereotaxic Surgery ........................................................... 190 
Amphetamine-mediated Rotational Behavior ....................................................... 190 
Levodopa Administration and LID Ratings ........................................................... 191 
Donor Tissue Preparation and Neural Cell Transplantation ................................. 192 
Graft-induced Dyskinesia (GID) ........................................................................... 193 
Necropsy .............................................................................................................. 194 
Histology .............................................................................................................. 194 
Tyrosine hydroxylase (TH) Immunohistochemistry for Stereological  
Quantification of Graft Cell Number and Volume ................................................. 194 
Stereological Quantification of Neurite Outgrowth ............................................... 195 
Immunofluorescence (IF) ..................................................................................... 196 
Fluorescent In Situ Hybridization (FISH) using RNAscopeTM ............................... 197 
Fluorescent Image Acquisition ............................................................................. 198 
Imaris® Fluorescent Image Quantification ........................................................... 199 
Statistical Analysis ................................................................................................ 201 
RESULTS ................................................................................................................ 203 
The homozygous rs6265 (Met/Met) genotype, in either host or donor, demonstrates 
superior graft efficacy and earlier amelioration of LID behavior ........................... 203 
Cell survival, graft volume, and neurite outgrowth are not significantly affected by 
the WT and/or homozygous rs6265 (Met/Met) genotype in host or donor ........... 208 
Homozygous rs6265 (Met/Met) parkinsonian rats engrafted with WT DA neurons 
remain the only host/donor combination to develop aberrant GID behavior ......... 210 
Homozygous rs6265 (Met/Met) parkinsonian rats engrafted with WT DA neurons 
express lower BDNF receptor transcript ratios (TrkB to p75NTR) .......................... 215 
Aberrant GID behavior in homozygous rs6265 (Met/Met) parkinsonian recipients of 
WT DA grafts is associated with excess DA release ............................................ 219 
GID behavior in homozygous rs6265 (Met/Met) parkinsonian rats engrafted with 
WT DA neurons is not correlated to immune marker expression in the parkinsonian 
striatum ................................................................................................................ 222 
DISCUSSION .......................................................................................................... 225 
BIBLIOGRAPHY ..................................................................................................... 237 

x 

 
 
CHAPTER 4: EXOGENOUS BDNF TREATMENT EXACERBATES GRAFT-INDUCED 
DYSKINESIA IN HOMOZYGOUS rs6265 (MET/MET) PARKINSONIAN RATS ........ 246 
ABSTRACT ............................................................................................................. 247 
INTRODUCTION ..................................................................................................... 248 
METHODS ............................................................................................................... 253 
Experimental Animals ........................................................................................... 253 
Experimental Timeline .......................................................................................... 254 
Nigrostriatal Lesioning with 6-OHDA .................................................................... 254 
Amphetamine-Induced Rotational Behavior ......................................................... 256 
Levodopa Administration and LID ratings ............................................................. 256 
Preparation of Donor Tissue and Cell Transplantation ......................................... 257 
Intrastriatal BDNF Infusions ................................................................................. 258 
Graft-induced Dyskinesia (GID) Ratings .............................................................. 259 
Necropsy .............................................................................................................. 259 
Histology .............................................................................................................. 260 
TH graft Cell Number and Volume ....................................................................... 260 
Neurite Outgrowth ................................................................................................ 261 
Immunofluorescence ............................................................................................ 262 
Fluorescent Image Acquisition ............................................................................. 262 
Imaris Fluorescent Image Quantification .............................................................. 264 
Statistical Analysis ................................................................................................ 266 
RESULTS ................................................................................................................ 268 

Exogenous BDNF infusion into DA-grafted animals enhances functional graft 
efficacy (i.e., amelioration of LID) and neurite outgrowth ..................................... 268 
Exogenous BDNF administration increased the severity and incidence of GID in 
DA-grafted homozygous rs6265 (Met/Met) rats ................................................... 273 
GID behavior is associated with behavioral and morphological indices of excess DA 
release in DA-grafted BDNF-infused animals ...................................................... 276 
Exogenous BDNF infusion increases microglial (Iba1) expression in DA-grafted 
animals ................................................................................................................. 285 
DISCUSSION .......................................................................................................... 290 
BIBLIOGRAPHY ..................................................................................................... 303 

CHAPTER 5: FUTURE DIRECTIONS AND CONCLUDING REMARKS ................... 314 
USING PRECISION MEDICINE TO GARNER MECHANISTIC INSIGHTS INTO GID 
BEHAVIOR .............................................................................................................. 316 
THE FUNCTIONAL BENEFIT OF rs6265 AND A POTENTIAL ROLE FOR THE 
BDNF PRO-PEPTIDE ............................................................................................. 321 
LIMITATIONS AND ALTERNATIVE APPROACHES .............................................. 326 
FUTURE DIRECTIONS ........................................................................................... 330 
The Benefit of the Met allele and the BDNF Met Pro-peptide .............................. 330 
Co-localization of VMAT2/VGLUT2 and Vesicular Synergy.................................. 331 
Graft Location....................................................................................................... 332 
Transplanting iPSCs into our rs6265 Parkinsonian Rat Model ............................. 334 
CONCLUDING REMARKS ..................................................................................... 336 
BIBLIOGRAPHY ..................................................................................................... 337 

xi 

 
LIST OF TABLES 

Table 1.1: Current Planned or Ongoing Clinical Trials for Cell Transplantation in PD. .. 52 

Table 3.1: Targeted Antigens with corresponding antibodies ....................................... 197 

Table 3.2: RNA Targets and RNAscopeTM probes ....................................................... 198 

Table 4.1: Targeted Antigens and corresponding antibodies ....................................... 263 

Table 5.1: Evidence of varied BDNF pro-peptide activity associated  
with rs6265 SNP expression. ...................................................................................... 323 

Table 5.2: Clinical Trials using iPSCs. ......................................................................... 335 

xii 

 
 
 
 
LIST OF FIGURES 

Figure 1.1: Progression time course of PD. ..................................................................... 5 

Figure 1.2: Classic Model of Basal Ganglia Circuitry in  
Normal & Parkinsonian brain. ........................................................................................ 11 

Figure 1.3: Risk Factors for PD. .................................................................................... 21 

Figure 1.4: Genetic variants in PD................................................................................. 25 

Figure 1.5: Sites of Action for Common Pharmacotherapies to treat PD. ...................... 35 

Figure 1.6: Types of Levodopa-induced dyskinesia (LID) and time course. .................. 37 

Figure 1.7: Unregulated Release of DA from a 5-HT Terminal. ..................................... 39 

Figure 1.8: Silencing of CaV1.3 Channels as a Promising Disease-Modifying Gene 
Therapy for PD. ............................................................................................................. 45 

Figure 1.9: Modeling Experimental GID in Rodents. ..................................................... 57 

Figure 1.10: hESCs vs. iPSCs as cell transplantation sources for PD. ......................... 72 

Figure 2.1: Precision medicine in Parkinson’s disease (PD) ....................................... 128 

Figure 2.2: BDNF Gene Structure, Processing, and Secretion. .................................. 135 

Figure 2.3: Schematic representations of conventional proBDNF and  
mBDNF signaling cascades. ....................................................................................... 140 

Figure 2.4: Summary of altered BDNF expression levels and consequences of the 
rs6265 SNP in neurodegenerative and psychiatric disorders ...................................... 144 

Figure 3.1: Experimental timeline and design. ............................................................ 189 

Figure 3.2: Impact of host/donor genotype on LID behavior and  
amphetamine-rotational asymmetry in DA-grafted parkinsonian rats .......................... 205 

Figure 3.3: Impact of host/donor genotype on graft survival and  
neurite outgrowth in DA-grafted parkinsonian rats. ..................................................... 209 

Figure 3.4: Impact of host/donor genotype on development of GID behavior and 
association with VGLUT2 expression. ......................................................................... 213 

xiii 

 
 
 
 
 
 
 
 
Figure 3.5: Impact of host/donor genotype on TrkB and p75NTR BDNF receptor 
transcript expression in DA-grafted parkinsonian rats. ................................................ 217 

Figure 3.6: Impact of host/donor genotype on DAT expression in DA-grated 
parkinsonian rats ......................................................................................................... 221 

Figure 3.7: Impact of host/donor genotype on immune marker (Iba1 and GFAP) 
expression in parkinsonian rats. .................................................................................. 223 

Figure 4.1: Experimental Design and Timeline ............................................................ 255 

Figure 4.2: Impact of BDNF supplementation on LID behavior  
and neurite outgrowth .................................................................................................. 270 

Figure 4.3: Impact of BDNF supplementation of GID behavior ................................... 275 

Figure 4.4: Exogenous BDNF administration is associated  
with indices of excess DA release ............................................................................... 280 

Figure 4.5: Exogenous BDNF infusion increases microglial (Iba1) expression in DA-
grafted animals. ........................................................................................................... 288 

Figure 4.6: Schematic diagram depicting the proposed mechanism of  
vesicular synergy. ........................................................................................................ 299 

Figure 5.1: A possible precision-medicine-based therapeutic approach  
to prevent and/or treat GID behavior prior or following DA cell transplantation. .......... 320 

Figure 5.2: Impact of the BDNF Met and WT pro-peptides on survival  
and volume (µm3) of TH+ DA neurons in cell culture. .................................................. 326 

Figure 5.3: Qualitative comparison of graft location and GID scores in  
each host/donor combination. ..................................................................................... 334 

xiv 

 
 
 
 
 
 
 
 
 
 
 
 
 
LIST OF ABBREVIATIONS 

2D 

3D  

Two-dimensional  

Three-dimensional 

3-OMD 

3-O-methyldopa 

5-HT   

5-hydroxytrypatmine (serotonin) 

6-OHDA  

6-hydroxydopamine  

8-OH-DAPT  8-Hydroxy-2-(di-n-propylamino)tetralin 

18F-DOPA 

Fluorodopa  

AADC  

Aromatic L-amino decarboxylase 

AAV 

AD 

Adeno-associated virus 

Alzheimer’s disease 

ADHD  

Attention deficit hyperactivity disorder 

AI 

Artificial Intelligence 

AIMs   

Abnormal involuntary movements 

ALS 

BBB 

Amyotrophic lateral sclerosis  

Blood-brain-barrier 

BDNF  

Brain-derived neurotrophic factor 

BG 

Basal ganglia  

CaV1.3 

Voltage-dependent, L-type calcium channel, alpha 1D subunit 

CMF   

Calcium-magnesium free  

CNS    

Central nervous system 

COMT  

Catechol-o-methyltransferase  

DA 

Dopamine 

xv 

 
 
 
 
 
 
 
 
 
 
DAB    

3,3'-Diaminobenzidine 

DAT 

Dopamine transporter 

DAT1   

Dopamine active transporter 1 gene 

DaTscan 

Dopamine transporter scan 

DBS 

DHF 

DJ-1 

DLB 

Deep brain stimulation 

7,8-Dihyrodxyflavone 

Parkinsonism-associated deglycase or Parkison disease protein 7 

Dementia with Lewy bodies 

dMSNs  

Direct pathway Medium Spiny Neuron 

DNA 

Deoxyribonucleic acid 

DREADD 

Designer Receptors Exclusively Activated by Designer Drugs 

DRD1  

Dopamine receptor 1 

DRD2  

Dopamine receptor 2 

DRT 

Dopamine replacement therapy 

ELLDOPA  Earlier vs. Later Levodopa Therapy in Parkinson’s disease 

EPA 

ER 

ERK 

Environmental Protection Agency 

Endoplasmic reticulum 

Extracellular signal-regulated kinase 

ESCs   

Embryonic stem cells 

eVM 

Embryonic ventral mesencephalic 

EWAS  

Epigenome-wide association study 

FBS 

FD 

Fetal bovine serum 

Fluorodopa 

xvi 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
FDA 

Food and Drug Administration 

GABA  

Gamma aminobutyric acid 

GBA 

Glucocerebrosidase A gene 

GDNF  

Glial-derived neurotrophic factor 

GFAP  

Glial fibrillary acidic protein  

GID 

Graft-induced dyskinesia  

GLP-1  

Glucagon-like peptide 1 

GPe 

GPi 

Globus pallidus externa 

Globus pallidus interna 

GSB-106   Bis-(N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide 

GWAS 

Genome-wide association study 

hAESCs 

Human amniotic epithelial stem cells 

HD 

Huntington’s disease 

hEVMs 

Human embryonic ventral mesencephalic cells 

hESCs 

Human embryonic stem cells 

hpNSC  

Human parthenogenetic neural stem cells 

Hz 

Iba1 

IHC 

Hertz 

Ionized calcium-binding adaptor molecule 1 

Immunohistochemistry 

iMSNs  

Indirect pathway medium spiny neuron 

iPSCs  

Induced pluripotent stem cells  

ISH 

i.p.  

In situ hybridization 

Intraperitoneal 

xvii 

 
 
 
 
 
 
 
 
 
 
 
 
 
JNKs   

c-Jun N-terminal kinases 

LAT1   

L-type amino acid transporter 1 

LB 

Lewy body 

L-DOPA 

Levodopa  

LN 

LID 

Lewy neurite 

Levodopa-induced dyskinesia 

LRRK2 

Leucine rich repeat kinase 2 

LTD 

LTP  

Long-term depression 

Long-term potentiation  

MAO   

Monoamine oxidase  

MAOBIs 

Monoamine oxidase type B inhibitors 

MAPK  

Mitogen-activated protein kinase 

MCI 

Mild cognitive impairment  

MDD   

Major depressive disorder 

MDS   

International Parkinson and Movement Disorder Society 

MFB 

Medial forebrain bundle 

MHC-II 

Major histocompatibility complex 2 

M/M 

Homozygous rs6265 Met/Met genotype grafted with Met/Met donor cells 

MMP   

Matrix metalloproteases  

MPTP  

1-Methyl,-4-phenyl-1,2,3,6-tetrahydropyridine 

mRNA  

Messenger ribonucleic acid 

MS 

Multiple sclerosis 

MSA   

Multiple system atrophy 

xviii 

 
 
 
 
 
 
 
 
 
MSCs  

Mesenchymal stem cells 

MSN   

Medium spiny neuron 

MTA 

Medial terminal nucleus 

mTOR  

Mechanistic target of rapamycin 

M/W 

Homozygous rs6265 Met/Met genotype engrafted with WT donor cells 

NeuN   

Pan neuronal marker 

NFκB   

Nuclear factor kappa B 

NGF 

Nerve growth factor 

NGS   

Normal goat serum 

NIH 

National Institute of Health 

NMDA  

N-methyl-D-aspartate  

NT-3   

Neurotrophin-3 

NT-4/5 

Neurotrophin-4/5 

OCD   

Obsessive compulsive disorder 

p75NTR 

pan 75 neurotrophin receptor 

PASCs 

Pluripotent stem cells isolated from adipose tissue 

PD 

Parkinson’s disease 

PDQ-39 

Parkinson’s disease questionnaire 39 

PET 

Positron emission tomography 

PI3K   

Phosphatidylinositol 3-kinase 

PINK1  

PTEN-induced putative kinase 1 

PLCγ   

Phospholipase Cγ 

PMI 

Precision Medicine Initiative 

xix 

 
 
 
 
 
 
 
 
 
PNS 

Peripheral nervous sytem 

PRKN  

Parkin gene 

PTSD   

Post-traumatic stress disorder  

PSP 

Progressive supranuclear palsy  

PWAS  

Pesticide-wide association study  

QSBB  

Queen Square Brain Bank 

rAAV   

Recombinant adeno-associated virus 

REM   

Rapid eye movement  

RhoA   

Ras homolog gene family member A 

RNA 

Ribonucleic acid 

s.c. 

Subcutaneous 

SERT  

Serotonin transporter 

shRNA 

Short hairpin ribonucleic acid 

SN 

Substantia nigra 

SNCA  

Synuclein alpha (α) 

SNpc   

Substantia nigra pars compacta 

SNpr   

Substantia nigra pars reticulata  

SNP 

Single nucleotide polymorphism 

SorCS2 

Sortilin-related Vps10p domain containing receptor 2 

SPECT  

Single photon emission computed tomography 

STN 

Subthalamic nucleus 

STR    

Striatum 

TBI 

Traumatic brain injury 

xx 

 
 
 
 
 
 
 
 
 
TBS 

Tris-buffered saline 

TBS-Tx  

Tris-buffered saline with Triton-X 

TGN 

Trans-Golgi network 

TH 

Trk 

TrkA 

TrkB 

TrkC 

Tyrosine hydroxylase 

Tropomyosin receptor kinase 

Tyrosine receptor kinase A 

Tyrosine receptor kinase B 

Tyrosine receptor kinase C 

UPDRS 

Unified Parkinson’s Disease Rating Scale 

US 

United States 

UTR 

Untranslated region 

VAChT 

vesicular acetylcholine transporter  

VGLUT 

Vesicular glutamate transporter 

VGLUT2 

Vesicular glutamate transporter 2 

VGLUT3 

Vesicular glutamate transporter 3 

VM 

Ventral mesencephalon 

VMAT2 

Vesicular monoamine transporter 2 

Vps10  

Vacuolar protein sorting 10 

VPS35   

Vacuolar protein sorting ortholog 35 

VTA 

W/M 

Ventral tegmental area 

WT genotype engrafted with Met/Met donor cells 

W/W    

WT genotype engrafted with WT donor cells 

xxi 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
CHAPTER 1: INTRODUCTION TO PARKINSON’S DISEASE (PD) 

1 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HISTORY 

In 1817, James Parkinson, an English surgeon and apothecary, was the first to 

describe the disease that came to bear his name, Parkinson’s disease (PD). He referred 

to the disorder as a shaking palsy or paralysis agitans. In his published work entitled, 

“An Essay on the Shaking Palsy,” he made prominent observations of individuals who 

demonstrated “involuntary tremulous motion, with lessened muscular power…with a 

propensity to bend the trunk forward, and to pass from a walking to a running pace; the 

senses and intellect being uninjured” (Parkinson, 2002). Those afflicted with the disease 

showed slow progression and a profound decrease in quality of life. Along with these 

symptoms, of which are now considered the classic motor symptoms of PD, Parkinson 

also remarkably noted the sleep and autonomic (e.g., constipation) components of PD, 

classified today as common non-motor features (Chaudhuri & Jenner, 2017; Goetz, 

2011; Parkinson, 2002).  

Fifty years following Dr. Parkinson’s observations, a French neurologist, Jean-

Martin Charcot, further described the manifestations of PD and distinguished 

bradykinesia as a primary motor feature. Charcot wisely recognized that not all 

individuals with PD demonstrated a marked weakness or tremor; therefore, he rejected 

the title of paralysis agitans or shaking palsy and recommended a name change to 

“Parkinson’s disease” (Charcot, 1892; Goetz, 2011). Many additional valuable 

observations were made in the years following, one of which was the identification that 

PD expressed a slight male predominance. This was discovered by William Gowers, a 

British neurologist, in 1888 (Gowers, 1898).  

2 

 
Although several clinical manifestations were detailed in regard to PD, it was not 

until the 1920s that significant pathological findings of the disease were determined. For 

example, Brissaud was the first to propose that damage to the substantia nigra (SN) 

may be the underlying pathology responsible for PD in 1925 (Edouard Brissaud, 1899). 

Also in the 1920s, additional pathological studies of the midbrain were separately 

conducted by Tretiakoff and Foix and Nicolesco (C Trétiakoff, 1921; Foix, 1925). The 

most comprehensive pathologic analysis which included the demarcation of brain 

lesions, however, was not performed until 1953 by Greenfield and Bosanquet (see 

(Greenfield & Bosanquet, 1953) for more details).  

Finally, in 1959, Bertler and Rosengren and Sano and colleagues proposed the 

possibility that dopamine (DA) was involved in the pathogenesis of PD. They 

demonstrated that the majority of DA in the brain was found in the caudate nucleus and 

putamen, both in dogs and in humans (Bertler & Rosengren, 1959; Hornykiewicz, 2010; 

Sano et al., 1959). To confirm, Oleh Hornykiewicz, an Austrian biochemist, analyzed the 

brains of patients with PD in 1960, discovering that these patients, indeed, had profound 

loss of DA in the caudate and putamen. Hornykiewicz further observed a loss of DA 

neurons in the SN, suggesting that this was the cause of the DA terminal loss in the 

striatum (Birkmayer & Hornykiewicz, 1961; Ehringer & Hornykiewicz, 1960). These 

findings enabled additional research to be conducted, specifically into the nigrostriatal 

pathway (Dahlstroem & Fuxe, 1964; Poirier & Sourkes, 1964; Sourkes & Poirier, 1965). 

The discovery of DA loss in these regions radically changed the field’s understanding of 

PD pathophysiology and remarkably led to the development of successful 

3 

 
pharmacotherapies (e.g., levodopa), some that remain clinically routine to this day 

(Cotzias et al., 1967). 

SYMPTOM PRESENTATION AND CLINICAL DIAGNOSIS 

Parkinson’s disease is the second most common neurodegenerative disease 

following Alzheimer’s disease (AD), affecting 9.3 million people worldwide (Espay et al., 

2017; Maserejian et al., 2020; Schalkamp et al., 2022). If PD maintains its current 

growth rate, approximately 13 million people are estimated to be diagnosed with PD by 

the end of 2040 (Dorsey et al., 2018; Straccia et al., 2022). Consequently, PD has 

placed significant strain on society. Not only does PD cause a poor quality of life, the 

total economic burden was estimated to be $51.9 billion in 2017 and projected to 

surpass $79 billion by the year 2037 (Yang et al., 2020). In the following paragraphs, the 

classic motor symptoms, non-motor symptoms, and clinical diagnosis of PD are 

discussed.  

Classic Motor Symptoms 

As a movement disorder, PD is known to exhibit several prominent motor 

features including bradykinesia, resting tremor, rigidity, and postural instability. The most 

characteristic of these symptoms is bradykinesia, which is defined as slowness of 

movement. While bradykinesia first manifests as a slowness in daily task performance 

(J. A. Cooper et al., 1994; Giovannoni et al., 1999), it often progresses to the loss of 

spontaneous movement, drooling (Bagheri et al., 1999), and a reduction in arm swing 

whilst walking (Jankovic, 2008). In addition to bradykinesia, another major symptom is 

resting tremor. These tremors occur at a frequency of 4-6 Hertz (Hz) and tend to affect 

the distal part of the extremities. While resting tremor does not often impact the neck, 

4 

 
head, or voice, it does involve the chin, jaw, and legs. Interestingly, resting tremor 

diminishes with action or while an individual is sleeping—a characteristic that helps 

differentiate PD from other disorders such as essential tremor (Jankovic, 2008).  

is  characterized  by  several  non-motor  symptoms 

Figure 1.1: Progression time course of PD. 
PD is often preceded by premotor symptoms (i.e., prodromal phase) of an estimated 20+ 
years.  This  phase 
including 
constipation, rapid eye movement disorder (REM), and depression. At time of diagnosis, 
when 50-60% of DA neurons in the substantia nigra (SNpc) have already been lost (Dauer 
&  Przedborski,  2003),  motor  symptoms  including  bradykinesia,  rigidity,  tremor,  and/or 
postural  instability  are  present.  Dyskinesias  (e.g.,  LIDs)  also  develop  in  approximately 
90% of patients by 10 years of treatment with levodopa (Hauser et al., 2017; Huot et al., 
2013,  2022). Abbreviations:  PD  =  Parkinson’s  disease.  REM  =  rapid  eye  movement. 
Schematic adapted from (L. V Kalia & Lang, 2015). 

Rigidity,  another  common  motor  feature,  is  characterized  by  stiffness  and 

increased  resistance  to  passive  movement.  Unfortunately,  rigidity  can  also  be 

accompanied by pain, often getting misdiagnosed as arthritis, bursitis, or a rotator cuff 

injury  (Jankovic,  2008;  Riley  et  al.,  1989).  The  last  major  motor  symptom  is  postural 

5 

 
 
instability, which tends to manifest in the later stages of PD. Postural instability involves 

the  loss  of  postural  reflexes,  frequently  resulting  in  falls  and  subsequent  hip  fractures 

(Williams, 2006). The later onset of falls, however, can be used to distinguish PD from 

other  parkinsonian  disorders including  multiple  system  atrophy  (MSA) and progressive 

supranuclear palsy (PSP). Although the discussed signs are considered to be the “classic 

hallmarks” of PD, there are additional secondary motor difficulties that several patients 

with  PD  can  also  exhibit.  These  include,  but  are  not  limited  to,  freezing  gait,  speech 

impairment  (e.g.,  microphonia),  micrographia  (small  handwriting),  and  respiratory 

disturbances (Figure 1.1) (Jankovic, 2008; Jankovic & Tolosa, 2007; Lees et al., 2009; 

Moustafa et al., 2016). 

Non-Motor Symptoms 

Despite being primarily considered a movement disorder, PD has long been 

associated with several non-motor signs and symptoms as well. Indeed, in James 

Parkinson’s report in 1817 (discussed previously), he observed non-motor symptoms 

alongside the classic motor symptoms in his patients (Parkinson, 2002). To date, 

numerous non-motor features have been noted in PD including constipation, urinary 

dysfunction, memory loss, depression, orthostatic hypotension, and sleep disturbances. 

Markedly, these non-motor symptoms frequently precede motor dysfunction by years or 

decades (Figure 1.1) (G. W. Ross et al., 2012; Tolosa et al., 2021). 

One of the most pronounced non-motor symptom individuals with PD exhibit is 

sleep disturbances, also referred to as rapid eye movement (REM) sleep behavior 

disorder. Over one-third of individuals will experience this disorder prior to their PD 

diagnosis, often mentioning an increase in violent dreams (Borek et al., 2007), as well 

6 

 
as sleep talking, kicking, and yelling. Also of note, greater than 50% of patients will have 

experienced insomnia to some degree (Boeve et al., 2007; Gjerstad et al., 2006). 

Additional non-motor features that patients with PD can demonstrate include obsessive 

compulsive disorder (OCD) and impulsive behaviors such as gambling or binge eating. 

Moreover, sensory abnormalities like olfactory dysfunction, a symptom not often 

recognized as a parkinsonian feature, recently have been correlated with a 10% 

increased risk of developing PD (Ponsen et al., 2004). Finally, although cognitive 

dysfunction is not yet fully understood in PD, a prospective study conducted by Aarsland 

and colleagues reported that patients with PD are at a sixfold increased risk for 

developing dementia (Aarsland et al., 2001).  

Clinical Diagnosis  

Currently, there is no definitive test to diagnose PD. Histopathological 

postmortem analysis is required to conclusively establish a PD diagnosis by confirming 

the presence of Lewy bodies (see Neuropathology below) (Jankovic, 2008). Although 

outside the realm of this thesis discussion, it is important to note that there are 

exceptions with specific genetic mutations (e.g., G2019S LRRK2) which lack Lewy body 

pathology at autopsy (see (O’Hara et al., 2020)). Nevertheless, there are clinical 

diagnostic criteria in place including the Queen Square Brain Bank (QSBB) criteria and 

the criteria generated by the International Parkinson and Movement Disorder Society 

(MDS), but these criteria are not without issue. For instance, in clinical practice, error 

rates for diagnostic misclassification can range from 15-24% (Hughes et al., 1992; 

Rajput & Rajput, 2014; Schrag, 2002; Tolosa et al., 2021). Furthermore, in over 10% of 

7 

 
cases that are diagnosed by PD neurologists, alternative pathologies were seen to be 

present upon postmortem autopsy (Tolosa et al., 2021).  

The criteria used for clinical PD diagnosis are centered around characterizing 

motor signs and symptoms. The QSBB criteria, which was initially proposed in the 

1980s, has been the most widely used criteria for clinical PD diagnosis up until 2015 

when the International Parkinson and MDS added its refinement (Gibb & Lees, 1988; 

Lees et al., 2009; Marsili et al., 2018; Postuma et al., 2015). Generally, these criteria 

rely on neurological examination, first identifying bradykinesia as a major motor 

symptom, in addition to resting tremor and/or rigidity (step one). Step two involves 

establishing that the patient does not exhibit symptoms or a history that would be 

indicative of another non-PD disorder (e.g., stepwise decline, repeated head trauma, 

encephalitis). Step three indicates whether the patient presents supportive criteria 

including resting tremor, unilateral onset, evidence of progression, a response to 

levodopa (L-3,4-dihydroxyphenylalanine), development of levodopa-induced dyskinesia 

(LID), and a long clinical course of 10 years or more (Gibb & Lees, 1988). 

In 2015, after scientific advancement, MDS refined the set of diagnostic criteria 

established by QSBB in order to further improve the diagnostic accuracy of PD. With the 

new criteria, two new diagnostic categories were created including the “Clinically 

Established PD” and “Clinically Probable PD” categories. These new categories 

incorporated what is referred to as “red flags:” factors that would exclude PD. However, 

when combined with supportive criteria, they do not exclude PD. Two ancillary tests 

were also added including olfactory dysfunction tests and cardiac imaging (Munhoz et 

al., 2024). Importantly, the MDS criteria has demonstrated great sensitivity (96%) and 

8 

 
specificity (95%) in a validation study for a clinical diagnosis of “probable PD” (Postuma 

et al., 2018). For individuals in the earlier stages of the disease (less than 5 years), the 

specificity of “clinically probable” PD was 87% (Postuma et al., 2018). In order to further 

enhance accuracy for early (i.e., prodromal) disease stages, additional tests and 

biomarkers are required.  

To reiterate, these criteria are based on motor signs and symptoms of PD. Yet, 

evidence has demonstrated that pathological and neurochemical markers for PD are 

established long before the exhibition of these motor symptoms. Because of this, and 

because non-motor symptoms are difficult to categorize so early-on due to their 

ambiguous nature, delineating non-motor, prodromal features of the disease (Figure 

1.1) will be imperative for the development of disease-prevention or disease-reversal 

therapy for PD. Scientists and clinicians have been making positive strides in this area: 

ancillary tests are being utilized concomitantly with the clinical diagnostic criteria in 

order to increase diagnostic accuracy. For example, molecular neuroimaging such as 

the dopamine transporter scan (DaTscan) can be used to discriminate between PD and 

essential tremor (Benamer et al., 2000). Unfortunately, however, DaTscan imaging 

cannot be used to differentiate between PD and MSA or PSP because of their shared 

degenerative characteristics (Tagare et al., 2017). Genetic testing has also been a 

useful ancillary test; however, only LRRK2 mutations have been screened successfully 

(Tolosa et al., 2006) and may be of limited value since over 90% of PD cases are 

idiopathic. Ultimately, it would be most promising if future research could uncover 

disease-specific biomarkers that would aid in the delineation between PD and other 

similar neurodegenerative disorders (Jankovic, 2008). 

9 

 
A correct diagnosis of PD is necessary for proper patient counseling and therapy 

development. Despite achieving rather high specificity and sensitivity, clinical diagnostic 

criteria remain fallible and still lead to misdiagnoses. While several ancillary tests have 

been developed and implemented in order to increase diagnostic accuracy, these tests 

are costly and not without caveats. Until further discoveries are made (e.g., biomarker 

development), histopathological confirmation of the presence of Lewy bodies in 

postmortem tissue remain the criteria for the definitive diagnosis of PD. The underlying 

neuropathology of PD is further discussed below.  

NEUROPATHOLOGY 

The Basal Ganglia 

The major pathological hallmarks of PD include the loss of DA neurons in the 

SNpc and the presentation of intracellular inclusion aggregates of the protein α-

synuclein, referred to as Lewy bodies (Obeso et al., 2002). Although these two features 

of PD are widely recognized as the pathological hallmarks of the disease, the underlying 

pathology is heterogenous and can vary greatly among individuals (Halliday et al., 

2008). Despite various other pathologies that may contribute to PD, the present 

discussion is only focused on these two neuropathological characteristics. The region of 

the brain that is most affected by these pathologies, subsequently leading to cardinal 

motor signs and symptoms of PD, is the basal ganglia (BG) (Figure 1.2a). Therefore, 

the structure and function of the BG are reviewed below. 

10 

 
a) 

b) 

Figure 1.2: Classic Model of Basal Ganglia Circuitry in Normal & Parkinsonian 
brain. 
(a) Coronal diagram of the BG in the human brain, excluding the substantia nigra pars 
compacta and reticulata, which are combined as SNpc in (b) for simplicity. (b) Schematic 
illustration  of  the  classic  BG  model,  including  the  direct  and  indirect  pathways  in  both 
healthy  and  parkinsonian  brains.  The  caudate  and  putamen  were  condensed  for 
simplicity. This is a limited representation of the mechanisms of the basal ganglia. Black 
lines indicate glutamatergic (solid) and GABAergic (dashed) neurons. The direct and  

11 

 
 
Figure 1.2 (cont’d) 
indirect  pathways  are  shown  in  green  and  red,  respectively.  Blue  arrows  demonstrate 
dopaminergic projections to both the direct (light blue) and indirect (dark blue) pathways. 
In the parkinsonian state, a red “X” demarcates the degeneration of the DA neurons in 
the substantia nigra pars compacta. Abbreviations: GPe = globus pallidus externa; GPi = 
globus  pallidus  interna;  SNpc  =  substantia  nigra  pars  compact;  STN  =  subthalamic 
nucleus. 

The  BG  are  a  group  of  seven  subcortical  nuclei  responsible  for  motor  control, 

reward-based learning, goal-directed behavior, and emotion (Chakravarthy et al., 2010; 

Lanciego et al., 2012). The seven nuclei include the caudate nucleus, putamen, globus 

pallidus  interna  (GPi),  globus  pallidus  externa  (GPe),  subthalamic  nucleus  (STN),  the 

substantia nigra pars compacta (SNpc), and the substantia nigra pars reticulata (SNpr) 

(Chakravarthy  et  al.,  2010).  This  list  can  further  be  categorized  into  input,  output,  or 

intrinsic  nuclei  (Lanciego  et  al.,  2012).  The  caudate  and  putamen  make  up  the  input 

nuclei, and functionally, these nuclei receive information from the cortex, the thalamus, 

and the SN. The output nuclei, including the GPi and the SNpr, send information to the 

thalamus. The GPe, the STN, and the SNpc are considered the intrinsic nuclei, and they 

relay  information  between  the  input  and  output  nuclei.  In-depth,  comprehensive 

summaries regarding BG anatomy can be found in (Chakravarthy et al., 2010; Gerfen & 

Wilson, 1996; MINK, 1996; Y. Smith et al., 1998). 

Functionally, the BG system requires the release of DA from SNpc neurons to its 

input nuclei (i.e., caudate and putamen), which are collectively called the striatum (STR) 

(Lanciego et al., 2012). Approximately 90% of the striatum consists of projection 

neurons (i.e., medium spiny neurons (MSNs)) and 10% interneurons. Structurally, MSNs 

are named for their appearance: they are multipolar neurons with medium-sized cell 

somas (~12-20µm in diameter), and their dendritic processes are covered with dendritic 

spines (Gerfen & Bolam, 2010). In general, two types of MSNs exist. Some MSNs 

12 

 
express dopaminergic receptor type 1 (DRD1), and some express dopaminergic 

receptor type 2 (DRD2), which generate two circuits that exert differential effects 

according to the classical model of the BG system. Both subtypes, however, release the 

inhibitory neurotransmitter, gamma aminobutyric acid (GABA; GABAergic) upon 

activation. 

The two circuits of the classical BG model include the direct and indirect 

pathways (Figure 1.2b). These circuits are thought to have oppositional effects (Albin et 

al., 1989; Calabresi et al., 2014; DeLong, 1990; Lanciego et al., 2012) in which the 

direct pathway proposedly promotes motor movement/selection, whereas activation of 

the indirect pathway is theorized to inhibit movement/selection. Neurons designated as 

A9 DA neurons from the SNpc project their axons onto the MSNs in the STR; the DA 

input on MSNs with DRD1 (dMSNs) exerts a faciliatory effect (direct pathway) and an 

inhibitory effect on DRD2-expressing MSNs (iMSNs; indirect pathway) (Chakravarthy et 

al., 2010; D. L. Clark et al., 2010; Lanciego et al., 2012). In this way, activation of the 

direct pathway will inhibit GPi activity, disinhibiting the thalamus, and promoting 

neuronal firing. The result is initiation of motor movement. Contrarily, activation of the 

indirect pathway will inhibit the activation of the GPe, disinhibit the STN, and allow for 

the GPi neurons to activate, inhibiting the thalamus, and ceasing motor movement. 

Under normal resting conditions, the indirect pathway is the “active” pathway in which 

tonically released DA inhibits activation of downstream motor systems (Chakravarthy et 

al., 2010). Upon phasic DA activation, the increase in striatal DA shifts the balance 

toward the direct pathway, allowing motor systems to activate (Chevalier et al., 1985; 

Deniau & Chevalier, 1985). 

13 

 
It is also important to note that glutamatergic excitatory projections from the 

cortex make synaptic connections generally onto the heads of dendritic spines of MSNs 

in the STR (Bouyer et al., 1984; Hattori et al., 1979; Z. C. Xu et al., 1989). 

Glutamatergic afferents from the thalamus similarly form connections onto MSNs; 

however, these afferents synapse onto the dendritic shafts of MSNs instead of the head 

of the spines (Dubé et al., 1988; Lacey et al., 2005; Z. C. Xu et al., 1991). The DA 

projections that extend from the SNpc make en passant synaptic appositions onto the 

necks of the dendritic spines of the MSNs and then modulate excitatory glutamatergic 

input coming into the MSNs from the heads of the same dendritic spines (Bamford et 

al., 2004; Bouyer et al., 1984; T. F. Freund et al., 1984; Gerfen & Surmeier, 2011; W. 

Shen et al., 2016; Yamamoto & Davy, 1992). In this way, both the glutamatergic input 

and the dopaminergic modulatory behavior is critical for normal motor function.  

In PD, the degeneration of DA neurons in the SNpc results in DA depletion in the 

STR (Figure 1.2b). Consequently, MSN activation in the direct pathway is reduced, 

resulting in a relative increase in the activity of the indirect circuit. The result is 

overstimulation of the GPi, ultimately diminishing movement execution, and thus leading 

to the classic motor features of PD. In the DA neurons that do survive, intracellular α-

synuclein inclusions tend to form, representing another pathology of PD. Both DA 

degeneration and α-synuclein aggregation pathologies are described below. 

Nigrostriatal Degeneration and DA Depletion 

One of the defining pathological characteristics of PD is the selective 

degeneration of dopaminergic neurons in the SNpc, specifically in the ventrolateral tier 

(A9) (Dickson, 2012; Fearnley & Lees, 1991; Kordower et al., 2013; Obeso et al., 2002; 

14 

 
Rudow et al., 2008). Interestingly, the dorsal and medial (i.e., A8 and A10) neurons are 

less vulnerable to degeneration, which has been demonstrated in both PD patients and 

animal models (Brooks et al., 1990; Iravani et al., 2005; Kish et al., 1988). From 

nigrostriatal afferents, DA is released tonically to the STR, with transient bursts of phasic 

release (A. A. Grace, 1991; A. Grace & Bunney, 1984). Rewarding events will induce 

brief phasic DA release, while adverse, negative events will decrease DA activity 

(Redgrave & Gurney, 2006; Schultz, 1998)—a phenomenon central to motor learning. 

As described above, at resting, tonic release of DA maintains sufficient DA levels in the 

STR as well as tonic DA receptor stimulation critical for normal BG function (Olanow et 

al., 2006; Venton et al., 2003). Degeneration of this system thus leads to a decrease in 

striatal DA, interfering with normal motor movement and action selection, thus resulting 

in motor symptoms of PD.  

The extent of nigrostriatal degeneration in individuals with PD has been studied. 

For example, Kordower and colleagues demonstrated that, at year one post-diagnosis, 

a modest loss of dopaminergic terminals in the STR was present visualized by 

decreased staining of dopaminergic markers (e.g. tyrosine hydroxylase (TH)). At three 

years post-diagnosis, there was marked loss of DA neuron staining (35-75%), and at 

four years, there was almost complete loss of DA fibers in the STR. Over the same time 

period and in the same patients, there was a 50-90% loss of DA neurons in the SNpc 

(Kordower et al., 2013). Further, other research groups have reported a 44-98% 

reduction in striatal DA levels in advanced PD (Bernheimer et al., 1973; Ehringer & 

Hornykiewicz, 1960; Rajput et al., 2008).  

15 

 
The morphological and functional effects of striatal DA depletion has also been 

studied extensively (see (Villalba & Smith, 2018) for review). More specifically, a 

significant reduction in spine density of striatal MSNs, both in length and in number 

(McNeill et al., 1988), has been observed in postmortem tissue from individuals with PD 

(Stephens et al., 2005; Villalba & Smith, 2018; Zaja-Milatovic et al., 2005), in 

parkinsonian rodent models (Ingham et al., 1989, 1998; Zhang et al., 2013), and in 

parkinsonian non-human primates (Villalba 2008). In addition to changes in spine 

density, glutamatergic reorganization has been observed in the DA-denervated striatum 

(Arbuthnott et al., 2000; M. Day et al., 2006; Gubellini et al., 2002; Ingham et al., 1998; 

Liang et al., 2008; Zhang et al., 2013). For instance, parkinsonian rodent models have 

exhibited a decrease in the quantity of glutamatergic asymmetric synaptic contacts onto 

MSNs in the striatum (Ingham et al., 1993, 1998). Collectively, this evidence 

demonstrates that DA plays a critical role in regulating the growth, maintenance, and 

plasticity of dendritic spines and glutamatergic connections onto MSNs (Arbuthnott et 

al., 2000; Robinson & Kolb, 1999). It remains to be determined, however, whether 

dendritic spine loss is an early or late-stage phenomenon in PD. Nevertheless, recent 

studies that targeted calcium channels in order to block calcium influx have exhibited 

promising prevention of dendritic spine loss in MSNs (Soderstrom et al., 2010; Steece‐

Collier et al., 2019); this could, in turn, be used as a therapeutic target to prevent the 

progression of DA degeneration in PD. 

Lewy Body Pathology 

The other defining pathological characteristic of PD is the accumulation of 

misfolded α-synuclein protein. Α-synuclein is a soluble, heat stable protein 

16 

 
approximately 140 amino acids in length (Jakes et al., 1994; McCann et al., 2014). Its 

physiological function remains elusive; however, several studies have proposed a role 

of α-synuclein in the maintenance of synapses, mitochondrial homeostasis, proteosome 

function, and DA metabolism (McCann et al., 2014; Ramalingam et al., 2023; Uversky, 

2003). Α-synuclein is known to be highly expressed in neurons of the frontal cortex, 

hippocampus, and the STR (Iwai et al., 1995; Norris et al., 2004). While under normal 

conditions, α-synuclein functions properly, in the context of PD, it forms insoluble 

inclusions within neuronal cell processes or cell bodies, referred to as Lewy neurites 

(LNs) and Lewy bodies (LBs), respectively.  

Frederick Lewy was the first to describe these α-synuclein LN/LB inclusions in 

1912. However, it was not until 1997-1998 that significant advancements were made 

that linked α-synuclein accumulation to LN/LB in PD and other disorders such as MSA 

or dementia with Lewy bodies (DLB) (McCann et al., 2014; Norris et al., 2004). Lewy 

bodies are described to contain a dense core of aggregated α-synuclein surrounded by 

a halo of fibrils that are approximately 10-15 nanometers (nm) in diameter (Arima et al., 

1998; Baba et al., 1998; Forno, 1969; Galloway et al., 1992; Spillantini et al., 1998; 

Tiller-Borcich & Forno, 1988). Indeed, cytoplasmic inclusions of α-synuclein are 

abnormal since α-synuclein is normally localized primarily to presynaptic terminals. 

Although the mechanistic consequences of Lewy pathology remain to be fully 

elucidated, it has been postulated that Lewy body inclusions negatively affect protein 

transport and organelle function (Duffy & Tennyson, 1965; Hill et al., 1991; M. L. 

Schmidt et al., 1991), often leading to cell death. In confirmation, experiments that have 

overexpressed α-synuclein in rodent models have demonstrated an inhibition of 

17 

 
neurotransmitter (i.e., DA) release (Gaugler et al., 2012; Nemani et al., 2010), as well as 

a 60-80% reduction of DA innervation to the STR (Lundblad et al., 2012). 

In 2003, Braak and colleagues generated a staging scheme for α-synuclein 

pathology largely based on the distribution and progression of α-synuclein over time 

(Braak et al., 2003). In this model, Lewy pathology is described in six stages. Pathology 

is first proposed to begin in the enteric nervous system and then travel to the dorsal 

motor nucleus of the vagus nerve in the medulla and to the olfactory nucleus (stage 1 

and 2) (Dickson, 2012). Pathology is then proposed to migrate to the locus coeruleus, 

and then to the DA neurons in the SNpc (stage 3). Later stages (i.e., 4-6) exhibit 

pathology in the basal forebrain, amygdala, and cortical areas (Dickson, 2012; McCann 

et al., 2014).  

While several clinical studies have reported results in favor of this Braak staging 

scheme (e.g., (Halliday et al., 2008)), one of which reported a 67% proportion of cases 

that successfully fit the staging scheme (Dickson et al., 2010), other groups have shown 

that pathology does not always follow the proposed distribution of α-synuclein. Indeed, 

in some elderly individuals with PD, Lewy pathology was exclusively found in the 

olfactory bulb (Beach et al., 2009; Fujishiro et al., 2008) or in the amygdala (Uchikado et 

al., 2006). Moreover, some neurologically “normal” individuals who were without PD 

signs or symptoms still exhibited widespread Lewy pathology (Frigerio et al., 2011; 

Parkkinen et al., 2005). Therefore, Braak staging remains a useful, but tentative, tool for 

PD pathophysiology. 

To date, there is controversy whether α-synuclein accumulation precedes 

neurodegeneration. While some argue that Lewy pathology is a precursor to neuronal 

18 

 
degeneration (Chu et al., 2024; Gibb & Lees, 1988), others have shown that, even at 

Braak stage 1 and 2, the quantity of DA neurons is already diminished (Milber et al., 

2012). Further challenging this central idea of α-synuclein accumulation toxicity, Lewy 

pathology is not always detected in PD brains (Buchman et al., 2012, 2019; L. V. Kalia 

et al., 2015; Milber et al., 2012; Yamashita et al., 2022), therefore suggesting that there 

could be an earlier, non-α-synuclein-related process involved in the degeneration of the 

nigrostriatal pathway in PD (Chu et al., 2024). Again, it is clear that PD is a 

heterogeneous and complex disorder as scientists continue to make strides in this field.  

RISK FACTORS AND ETIOLOGY 

Parkinson’s disease is a complex and multifaceted neurodegenerative disorder of 

largely unknown etiology. Currently, a combination of genetic and environmental risk 

factors are thought to contribute to the development of the disorder. Several 

demographic characteristics have also been associated with PD risk including gender, 

ethnicity, and advancing age, with age being the greatest risk factor (Collier et al., 2011, 

2017; L. V. Kalia et al., 2015; Van Den Eeden, 2003). In addition to age, environmental 

factors such as toxicant exposure, and genetic susceptibilities, research continues to 

identify other elements that may influence the likelihood of developing PD. For instance, 

traumatic brain injury (TBI), lifestyle choices such as diet and exercise, and diabetes 

have been more recently reported as possible risk factors or comorbidities of PD 

(Figure 1.3).  

Advancing Age 

Advancing age is known to be the greatest risk factor for developing PD (Bennett 

et al., 1996; Collier et al., 2011; J. F. Cooper et al., 2015; Morens et al., 1996; Tanner & 

19 

 
Goldman, 1996; Wyss-Coray, 2016). Yet, advancing age as a risk factor is not specific 

to PD: it is common in many other neurodegenerative diseases such as AD. Indeed, it is 

estimated that one in ten individuals over the age of 65 currently has AD, a prevalence 

that will continue to rise as our aging population increases (Hou et al., 2019). The aging 

US population (≥65 years) is estimated to increase to 88 million in the year 2050 (from 

53 million in 2018) (Hou et al., 2019). Therefore, the burden of PD will continue to 

expand, and identifying ways to halt or slow its progression continues to be a priority in 

the field of neurodegeneration. 

The role of aging in PD pathogenesis remains elusive (Pang et al., 2019). 

However, the hallmarks of aging, some of which include genome instability, telomere 

degradation, epigenetic alterations, loss of proteostasis, and mitochondrial dysfunction, 

share important biological features with PD and have been correlated to an increased 

PD risk (for review (Hou et al., 2019)). The overlap of the molecular mechanisms of 

aging and PD continue to allow scientists to make strides in neurodegenerative 

research. Experimentally, postmortem analyses of neurologically “normal” brains of 

individuals between the ages of 14 and 92 years old have established a significant 

decrease of striatal DA with advancing age (Kish et al., 1992). A loss of brain weight and 

SN volume have also been demonstrated in aging humans and non-human primates (E. 

Y. Chen et al., 2000; Chu et al., 2002). Aging mechanisms continue to be a target for 

potential therapies in PD and for other neurodegenerative disorders. 

20 

 
Figure 1.3: Risk Factors for PD.  
Schematic representation of risk factors associated with developing PD. While not fully 
elucidated, a combination and/or interaction of risk factors is thought to contribute to the 
incidence of PD. The factors presented here are only examples and are not a complete 
list. Abbreviations: TBI = traumatic brain injury.  

21 

 
 
 
Genetic Risk Factors 

Approximately 5-10% of patients with PD exhibit a monogenic (i.e., caused by a 

mutation in a single gene) form of the disorder. As of 2020, over one hundred 

pathogenic risk loci in PD have been identified using genome-wide association studies 

(GWAS) (Blauwendraat et al., 2020). Defined below, the major autosomal dominant 

mutations that have been identified include SNCA, LRRK2, and VPS35, whereas the 

autosomal recessive mutations are found in PINK1, DJ-1, and Parkin, all of which are 

known to cause PD with high penetrance. Several other genes with Mendelian 

inheritance also have been implicated in PD, specifically atypical PD (Lill, 2016; Lunati 

et al., 2018), but are not as prevalent in the general population. Overall, the vast 

majority of PD is extraordinarily complex, and it is more likely that PD is caused by a 

combination of genetic and environmental risk factors. 

SNCA 

The first autosomal dominant mutation associated with PD was found in the 

SNCA gene in 1997 (Polymeropoulos et al., 1997). SNCA encodes for α-synuclein, and 

mutations in SNCA tend to cause abnormal α-synuclein accumulation, leading to LB and 

LN formation (see Neuropathology section). Moreover, missense mutations or 

duplications of SNCA produce signs of dementia in patients with PD (Lill, 2016). 

Interestingly, there is a dosage effect of mutations in this gene. For instance, 

triplications, compared to duplications, can induce an earlier age of onset of PD of 

which progresses more rapidly (Lill, 2016; Lunati et al., 2018).  

22 

 
LRRK2 

Leucine-rich repeat kinase 2 (LRRK2) is another autosomal dominant mutation 

that has been implicated in the risk of PD development. To date, nine highly penetrant, 

pathogenic mutations have been found in LRRK2 (Healy et al., 2008; Paisán-Ruiz et al., 

2013; O. A. Ross et al., 2011; Rubio et al., 2012). LRRK2 encodes for a protein called 

dardarin which is involved in lysosomal and autophagy regulation (Lunati et al., 2018). 

Consequently, mutations in LRRK2 lead to the hyperactivation of its kinase domain 

(Alessi & Sammler, 2018); therefore, potential LRRK2 antagonists are currently being 

studied as a therapeutic for this genetic form of PD. Similar to SNCA mutations, as well 

as idiopathic PD, individuals with LRRK2 mutations exhibit typical PD symptoms and 

respond well to levodopa.  

VPS35 

The third major autosomal dominant mutation associated with PD risk is found in 

the gene for vacuolar protein sorting 35 (VPS35). This gene encodes for a protein 

responsible for synaptic endocytosis and retrograde protein transport. In this way, 

mutations in VPS35 are postulated to disrupt vesicle formation and protein trafficking 

(Lunati et al., 2018; Trinh & Farrer, 2013). PD patients with VPS35 mutations 

demonstrate typical PD symptoms and a good response to levodopa, like that of 

patients with the SNCA and LRRK2 gene mutations.  

GBA 

The most prominent genetic risk factor for PD is found in the glucocerebrosidase 

A gene (GBA). GBA encodes for glucocerebrosidase, a lysosomal hydrolase enzyme 

that catalyzes the breakdown of both glucosylceramide and glucosylsphingosine 

23 

 
(Sidransky & Lopez, 2012; L. Smith & Schapira, 2022). Approximately 5-15% of 

individuals with PD have GBA mutations, occurring more frequently than any other gene 

in familial PD (e.g., SNCA, LRRK2) (Sidransky et al., 2009), and well over 300 

pathogenic GBA mutations have been identified to date (Beutler et al., 2004; Hruska et 

al., 2008). Those with GBA mutations have an average age of onset that is estimated to 

be five years earlier than idiopathic PD (Gan-Or et al., 2008; Malek et al., 2018; 

Neumann et al., 2009; Sidransky et al., 2009), and their risk of developing cognitive 

deficits and dementia is also greater (Cilia et al., 2016; Papapetropoulos et al., 2006; 

Petrucci et al., 2020). Functionally, homozygous GBA mutations have been described 

as causative factors for Gaucher’s disease, which is a lysosomal storage disorder (see 

Figure 1.4). 

24 

 
 
Figure 1.4: Genetic variants in PD.  
grouped  based  on  allelic  frequency  and  penetrance.  Autosomal  dominant  genes  are 
labeled blue, and autosomal recessive genes are labeled in green. Risk loci are labeled 
gray. Adapted from (J. O. Day & Mullin, 2021; Gasser, 2015).  

Parkin, PINK1, and PARK7 

Autosomal recessive mutations have also been linked to the risk of developing 

PD. The significant at-risk genes that have currently been mapped include PRKN 

(Parkin) (Kitada et al., 1998), PINK1 (Valente et al., 2004), and PARK7 (DJ-1) (Bonifati 

et al., 2003). The most common of these is Parkin, which accounts for 8.6% of early-

onset (<50 years) PD; PINK1 accounts for 3.7%, and DJ-1 accounts for 0.4% (Abou‐

Sleiman et al., 2003; Kilarski et al., 2012). Parkin specifically encodes for E3 ubiquitin 

ligases, which are enzymes that are responsible for the degradation of damaged 

25 

 
 
proteins (Shimura et al., 2000; K. Tanaka et al., 2001)). Therefore, mutations in Parkin 

(and PINK1) are thought to be associated with lysosomal degradation dysfunction (Deliz 

et al., 2024). In contrast, mutations in DJ-1 cause deficits in protecting neurons from 

oxidative stress (Kim et al., 2005). Like the major autosomal-dominant mutations, these 

autosomal recessive mutations (Parkin, PINK1, and DJ-1) all exhibit typical signs and 

symptoms of PD. However, despite sharing a similar phenotype, those with DJ-1 

mutations tend to have more non-motor symptoms including depression, psychosis, and 

cognitive deficits when compared to those with Parkin and PINK1 mutations (Kasten et 

al., 2018; Kilarski et al., 2012). 

Various other autosomal dominant and recessive mutations implicated in PD risk 

exist; however, they are outside the scope of this dissertation. Furthermore, it is 

important to note that the field of epigenetics has been, and continues to be, extensively 

studied in PD. Epigenetics involves the chemical modification (e.g., methylation) of 

DNA, resulting in alteration of gene expression. Despite the importance of epigenetics in 

PD, scientists have yet to conduct an epigenome-wide association study (EWAS) for PD 

(Lill, 2016). 

Environmental Risk Factors 

In addition to genetic risk factors of PD, several environmental toxicants have 

been identified as key risk factors for PD. In 2023, Paul and colleagues conducted a 

pesticide-wide association study (PWAS). From this study, 39 common pesticides were 

found to be associated with PD risk, the majority of which are known to induce 

dopaminergic cell death (Paul et al., 2023). Some of these chemicals/pesticides 

26 

 
associated with increased PD incidence include paraquat, rotenone, cyanide, dieldrin, 

and manganese (Di Monte et al., 2002; Gorell et al., 1998; Monte, 2003). 

MPTP 

The idea that contact with various pesticides could increase the risk of PD first 

came from the observation of 1-methyl,-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 

exposure. MPTP, which shares structural similarity to the herbicide paraquat (Ball et al., 

2019; Kanthasamy et al., 2005; Langston et al., 1983), induced “textbook-like” signs of 

advanced PD in a small group of drug addicts in 1983 (Ball et al., 2019; Kanthasamy et 

al., 2005; Langston, 1998; Langston et al., 1983). The “textbook-like” symptoms of PD 

became understandable upon the discovery that MPTP exposure was determined to 

induce mitochondrial toxicity in dopaminergic neurons of the SNpc (Chaturvedi & Flint 

Beal, 2013). This discovery catalyzed additional investigations into other 

chemicals/pesticides to determine whether their exposures could also be associated 

with increased incidence of idiopathic PD. 

Paraquat 

Moving forward from MPTP, scientists began investigating paraquat, again 

because of its structural similarity to MPTP. Paraquat exposure in individuals has been 

reported to induce DA neuron cell death, α-synuclein aggregation, and 

neuroinflammation (Pouchieu et al., 2018; Purisai et al., 2007; Richardson et al., 2007). 

Likewise, in a rodent model, paraquat administration killed off DA neurons in the SNpc 

in a dose- and age-dependent manner (McCormack et al., 2002). Consequently, 

paraquat has been definitively linked to increasing the risk for PD (Kitazawa et al., 

27 

 
2003). Indeed, in the Agricultural Health study of 110 PD patients, a positive association 

was discovered between risk of PD and exposure of paraquat (Tanner et al., 2011).  

Rotenone 

Another at-risk pesticide for PD development is rotenone, a major 

organophosphate pesticide frequently used in the control of fish populations (Betarbet et 

al., 2000; Tanner et al., 2011). Mechanistically, rotenone, similar to MPTP, is 

characterized as a selective inhibitor of the mitochondrial complex I; it is also well-

known to promote and accelerate the aggregation of α-synuclein (Silva et al., 2013; 

Yuan et al., 2015). PD cases have been linked to chronic rotenone exposure in 

epidemiological studies (Dhillon et al., 2008; Tanner et al., 2011). Particularly, in a 

French AGRICAN study, an increased risk of PD was reported in farmers who were 

exposed to rotenone (Pouchieu et al., 2018). 

Dieldrin 

PD has also been known to be caused by an organochlorine pesticide referred to 

as dieldrin. In the 1970s, Dieldrin was widely used as an insecticide; however, in 1974, 

the United States Environmental Protection Agency (US EPA) banned its use due to its 

propensity for bioaccumulation and its potential carcinogenic effects (Kanthasamy et al., 

2005). Several animal models have demonstrated the detrimental effect of dieldrin on 

the dopaminergic system (Kanthasamy et al., 2005). For example, rodent models have 

confirmed the targeting of dieldrin to the DA system in a dose-dependent manner 

(Hatcher et al., 2007; Richardson et al., 2006). Also, in ring doves, significant depletion 

of DA levels (58.6%) in the brain was reported in response to low-dose dieldrin 

exposure (Heinz et al., 1980). Most importantly, in postmortem human PD brain tissue, 

28 

 
dieldrin exposure was found to induce cell death in the SNpc (Corrigan et al., 1998; 

Miller et al., 1999). 

Other Risk Factors and Comorbidities 

Over the past decades, TBI has emerged as a possible risk factor for developing 

PD. TBI is known to cause breakdown of the blood-brain-barrier (BBB), as well as 

chronic inflammation, mitochondrial dysfunction, and α-synuclein accumulation (Marras 

et al., 2014). This has been confirmed in rodent models of TBI in which the animals 

developed α-synuclein aggregation and DA cell loss in the SNpc (Acosta et al., 2015). 

Behaviorally, rodents with TBI exhibited PD-like behavior at six months after injury (Sha 

et al., 2025). Moreover, individuals with a history of head trauma were at a higher risk of 

developing PD (Jafari et al., 2013). It must be considered, however, that there is an 

overall 50% increase in falls/head injuries approximately three months prior to a PD 

diagnosis; therefore, a correlation/causation of TBI and PD cannot yet be 100% 

corroborated. Indeed, one study found that there was no association between a TBI 

experienced 10 or more years prior to a PD diagnosis (Kenborg et al., 2015).  

Metabolic syndromes (e.g., diabetes) have recently elicited increased interest as 

a possible risk factor and/or comorbidity of PD (Chohan et al., 2021; Cullinane et al., 

2023; Leibson et al., 2006). It has been postulated that these metabolic syndromes 

have similar cellular mechanisms, such as mitochondrial dysfunction, to that of PD. A 

significant increase in the risk of developing PD has been documented in individuals 

with type 2 diabetes in reports from Finland (Hu et al., 2007), Denmark (Schernhammer 

et al., 2011), Taiwan (Sun et al., 2012), the Physician health study (Driver et al., 2008), 

and NIH-AARP (Q. Xu et al., 2011). Correlations between type 2 diabetes and an 

29 

 
increased severity of motor and non-motor symptoms at the time of PD onset has also 

been noted (Athauda et al., 2022). In contrast, however, this phenomenon was not seen 

in two large US cohort studies (Palacios et al., 2011; Simon et al., 2007). Despite these 

findings, pharmaceuticals that treat diabetes have been found to induce neuroprotective 

effects in PD models (Santiago et al., 2017). Further research in these areas (e.g., TBI, 

diabetes) is warranted in order to confirm a relationship with PD risk as well as its 

underlying mechanisms.  

In addition to an extensive list of risk factors that increase the incidence of PD, 

there have been studies that have also revealed possible protective factors that may 

lower PD risk. Some of these include smoking, caffeine consumption, ibuprofen use, 

and physical activity (Ascherio & Schwarzschild, 2016; Noyce et al., 2012). While these 

analyses may seem promising, some of these factors could be contentious (e.g., 

smoking), and thus require further research to definitively determine whether these 

habits truly lower the risk of PD. Exercise, as a beneficial example, has been 

epidemiologically associated with a reduced risk of PD (H. Chen et al., 2005; Thacker et 

al., 2008), and therefore, may be an up-and-coming, widely-prescribed therapeutic 

approach to treat PD. Additional therapeutic strategies will be discussed in the next 

section.  

30 

 
 
 
THERAPEUTIC STRATEGIES FOR PD 

Pharmacotherapy 

The current pharmacological treatments for PD include DA replacement 

therapies (DRTs) and advanced surgical therapies such as deep brain stimulation (DBS) 

in the event that DRT becomes difficult to manage due to fluctuating responses. While 

DRTs are mostly successful in treating the motor symptoms of PD, they unfortunately do 

little to treat the non-motor symptoms. Moreover, no interventions currently exist that 

can prevent, delay, or reverse disease progression (i.e., disease-modifying therapies) 

(Fahn, 2003; Fox et al., 2018; Noyce et al., 2016; Poortvliet et al., 2020). Because gaps 

in our understanding of the underlying cause of PD remain, it is difficult to create 

treatments that will modify the pathology of PD (Lang & Espay, 2018). Nevertheless, in 

addition to optimizing currently available symptomatic treatments, researchers 

continuously endeavor to discover therapies directed at disease modification; several 

pathways are being investigated as potential treatment targets. This section will explore 

the first-line DRTs, advanced therapies, and experimental (potentially disease-

modifying) treatments currently available for PD.  

Levodopa (L-DOPA) was first isolated in 1910 by Torquato Torquati, but it was not 

until 1957 that its connection to DA and PD were discovered (A. Carlsson et al., 1957; 

Hornykiewicz, 2010). In 1957, Arvid Carlsson, a Swedish pharmacologist, remarkably 

demonstrated that levodopa diminished parkinsonian symptoms in reserpine-treated 

mice and rabbits. Reserpine, an alkaloid that blocks monoamine transport (A. Carlsson 

et al., 1957), induces a tranquilizing, parkinsonian-like state, and therefore was a useful 

model at the time for these studies (A. Carlsson et al., 1957). Within 15-30 minutes of 

31 

 
 
levodopa administration to the reserpine-treated animals, mice and rabbits returned to 

almost-normal behavior, ameliorating their parkinsonian state. However, the effect of 

levodopa only lasted for an hour, and animals returned back to their reserpine-induced 

parkinsonian-like state (A. Carlsson et al., 1957). Despite dose/timing caveats, these 

preliminary experiments demonstrated considerable potential for the use of levodopa in 

the treatment of PD.  

A year later, Carlsson’s research group determined that DA content in the brain 

increased upon levodopa administration, initiating their postulations of DA being 

implicated in motor disorders (e.g., PD). Then, in 1960, Ehringer and Hornykiewicz 

reported that patients with PD exhibited caudate and putaminal DA depletion (Ehringer 

& Hornykiewicz, 1960; Fahn, 2008). Following up on this observation, Hornykiewicz and 

Birkmayer intravenously administered levodopa to patients with PD, notably 

demonstrating distinct alleviation of their motor symptoms (Birkmayer & Hornykiewicz, 

1961; Fahn, 2015). However, levodopa-infused patients developed distressing 

gastrointestinal-upset in response to the high doses of levodopa. To remedy this 

problem, Cotzias and colleagues decided to slowly increase the dose overtime, 

successfully avoiding gastrointestinal side effects (Cotzias et al., 1967). 

Today, almost 65 years later, levodopa remains the most effective 

pharmacological intervention for PD (Cotzias et al., 1967; Poewe et al., 2010; Stoker & 

Barker, 2020). Compared to other DRTs (discussed below), levodopa demonstrates 

superior motor improvement when assessed by reductions in the United Parkinson’s 

Disease Rating Scale (UPDRS) scores (Poewe et al., 2010). Importantly, levodopa is 

also better tolerated than DA agonists, especially in elderly patients (>60 years of age) 

32 

 
(Nutt & Wooten, 2005; Poewe et al., 2010). Mechanistically, in the presence of a 

peripheral decarboxylase inhibitor, levodopa crosses the BBB and is converted to DA by 

aromatic L-amino acid decarboxylase (AADC), an enzyme found in catecholaminergic 

neurons. Following its release into the synapse, DA will then be metabolized by 

catechol-O-methyltransferase (COMT) or monoamine oxidase (MAO) (Figure 1.5a) 

(National Institute of Diabetes and Digestive and Kidney Diseases, 2012a). Because of 

its short half-life (≤ 90 minutes) (Poewe et al., 2010) and its mechanism of action, 

levodopa is given in conjunction with carbidopa (an L-amino acid decarboxylase 

inhibitor) to prevent is metabolism in the periphery; COMT and MAO inhibitors can also 

be given alongside levodopa to prolong its half-life in the body (Fahn, 2003).  

The success of levodopa can be limited by side effects than can manifest 

following chronic levodopa administration and continued progression of PD. Like other 

DRTs, some side effects of levodopa therapy can include nausea, hallucinations, 

confusion, postural hypotension, constipation, depression, and sleep disturbances 

(Bastide et al., 2015; National Institute of Diabetes and Digestive and Kidney Diseases, 

2012a). The most bothersome and detrimental side effect, however, is the development 

of levodopa-induced dyskinesia (LID) (Poewe et al., 2010) (see Long-term side effects 

of Chronic Levodopa Therapy). 

Often less tolerated and less effective than levodopa therapy (Stoker & Barker, 

2020) are DA agonists, DA metabolism inhibitors (i.e., monoamine oxidase inhibitors; 

MAOIs), anticholinergics, adenosine antagonists, and β-blockers, all of which are 

available as additional medications used to treat the motor symptoms of PD (Figure 

1.5b). Even though levodopa is considered the gold standard, sometimes other DRTs 

33 

 
will be prescribed first, frequently in younger patients (<60 years old), to avoid/delay the 

side effect of LIDs (Connolly & Lang, 2014). Apomorphine is an example of a DA 

agonist currently available on the market. Agonists specific to the D2 receptor include 

ropinirole, pramipexole, and rotigotine (National Institute of Diabetes and Digestive and 

Kidney Diseases, 2012b). COMT inhibitors have also been developed to block the 

breakdown of DA by catechol-O-methyltransferase (COMT). COMTs, as well as MAOIs, 

can be used in conjunction with DA agonists or levodopa to help manage PD symptoms 

(National Institute of Diabetes and Digestive and Kidney Diseases, 2012b). 

Nevertheless, compared to levodopa, DA agonists and MAOIs/COMTs are more likely to 

induce side effects such as hallucinations, psychosis, compulsive behaviors, sleep 

disturbances, nausea, and confusion, especially in elderly patients (Armstrong & Okun, 

2020; Bloem et al., 2021; Fahn, 2003), and therefore, levodopa is often preferred 

regardless of the risk of LID development. 

34 

 
 
 
Figure 1.5: Sites of Action for Common Pharmacotherapies to treat PD.  
Schematic diagram depicting the sites of action for various PD medications used to treat 
the  motor  symptoms  of  PD.  (a)  Levodopa  is  converted  to  dopamine  (DA)  by  aromatic 
amino acid decarboxylase (AADC) both in the circulatory system and in the brain. Dopa  

35 

 
 
Figure 1.5 (cont’d) 
decarboxylase  inhibitors  (DDCIs)  are  used  to  prevent  levodopa’s  conversion  in  the 
periphery,  allowing  higher  concentrations  of  levodopa  across  the  blood  brain  barrier 
(BBB). In the striatum, nigrostriatal dopaminergic afferents, corticostriatal glutamatergic 
afferents, and cholinergic interneurons converge to regulate the activity of medium spiny 
GABAergic neurons  (MSNs).  Once  levodopa  is converted  into  DA  here  in  the  striatum 
inside DA terminals, replacing the neurotransmitter deficit in PD, DA will bind and activate 
DA receptors (D1 and D2) on the resident striatal MSNs, permitting motor movement. (b) 
Likewise,  DA  agonists  and  MAOIs  also  restore  motor  function  as  they  activate  DA 
receptors  and  prevent  DA  degradation,  respectively.  Other  therapeutics  such  as 
amantadine (pink) inhibit the activity of N-methyl-D-aspartate (NMDA) receptors to treat 
dyskinesias. Anticholinergics are another pharmacologic; they are used to treat tremors 
by  blocking  nicotinic  acetylcholine  receptors  (blue).  Figure  has  been  adapted  from 
(Connolly  &  Lang,  2014).  Abbreviations:  3-OMD  =  3-O-methyldopa;  DDCIs  =  dopa 
decarboxylase  inhibitors;  AADC  =  aromatic  amino  acid  decarboxylase;  COMTs  = 
catechol-O-methyltransferase inhibitors; DA = dopamine; MAOIs = monoamine oxidase 
inhibitors; LAT1 = L-type amino acid transporter 1 

Long-term Side Effects of Chronic Levodopa Therapy 

LIDs are characterized as abnormal involuntary movements in response to 

chronic levodopa therapy (Poewe et al., 2010). These abnormal movements tend to 

affect the neck, upper limbs, and torso, triggering chorea, ballism, dystonia, and 

myoclonus (Kwon et al., 2022; Vijayakumar & Jankovic, 2016), all of which can cause 

substantial discomfort in individuals with PD (Hung et al., 2010; Khlebtovsky et al., 

2012; Prashanth et al., 2011). Estimates of the incidence of LID vary by source but 

relatively reflect that approximately 50% of patients will develop LID during the first 3-5 

years of levodopa treatment (Blanchet et al., 1996; Manson et al., 2012). By 10-15 

years of treatment, 50-94% of patients exhibit LID (Ahlskog & Muenter, 2001; Fahn, 

2003; Hely et al., 2005; Stoker & Barker, 2020). Moreover, in a large retrospective study 

referred to as the ELLDOPA study (Earlier vs. Later Levodopa therapy in PD), patients 

exhibited variable motor improvement ranging from 100% improvement to 242% 

worsening of symptoms (Hauser et al., 2009).  

36 

 
Figure 1.6: Types of Levodopa-induced dyskinesia (LID) and time course.  
After  a  single  dose  of  levodopa,  the  supratherapeutic  window  is  reached  and  is 
characterized by peak-dose dyskinesias. Following, the therapeutic window is considered 
the  ON-state  when  optimal  clinical  benefit  is  reached  with  no  dyskinetic  behavior. 
Diphasic (or biphasic) dyskinesias will appear in the transitional window, during the rise 
and  fall  of  levodopa  levels  in  the  plasma.  In  the  OFF-state,  subtherapeutic  window, 
parkinsonian symptoms are prevalent again as the effects of levodopa are lost. Adapted 
from  (di  Biase  et  al.,  2023).  Abbreviations:  [levodopa]  =  plasma  concentration  of 
levodopa.  

The two most common forms of dyskinesia include peak-dose dyskinesia and 

diphasic dyskinesia (Figure 1.6). Peak-dose dyskinesia is the most prominent with 75-

80% of patients experiencing this type of dyskinesia (Zesiewicz et al., 2007). Peak-dose 

dyskinesia occurs when plasma levels of levodopa are at their highest. Choreiform 

movements dominate in this form, but other movements including dystonia, myoclonus, 

37 

 
 
and ballism in the orofacial muscles also occur (Vijayakumar & Jankovic, 2016). In 

contrast to peak-dose dyskinesia, diphasic dyskinesia appears when plasma 

concentrations of levodopa are rising and falling (Zesiewicz et al., 2007). Specifically, 

diphasic dyskinesias manifest when levodopa is first given and when levodopa begins 

to wear off. Dystonic or ballistic movements most often characterize this form (Rascol et 

al., 2001). 

While the underlying mechanisms of LID are unclear, current research suggests 

that LIDs manifest due to the non-physiological DA release and activation of striatal DA 

receptors induced by pharmacological administration of levodopa. To attempt to 

diminish LID behavior, pharmacologists are investigating various other formulations of 

levodopa to improve its delivery and its half-life (Poewe et al., 2010). Although LIDs can 

significantly impact quality of life, it is often said that individuals generally prefer their 

dyskinetic movements compared to limited movement with PD motor symptoms 

(Khlebtovsky et al., 2012), however, see (Cenci et al., 2020).  

Several underlying mechanisms of LID have been postulated, one of which being 

the serotonin theory. Briefly, serotonin (5-HT) neurons have the ability to take up and 

convert exogenous levodopa into DA, but they lack the machinery to reuptake released 

DA and provide autoreceptor-mediated feedback to the neuron (Figure 1.7). Therefore, 

it is theorized that 5-HT neurons can cause excessive, non-physiological stimulation of 

the striatal DA receptors, thus resulting in LID (Bezard, 2013; Sellnow et al., 2019). 

Studies in favor of this theory have utilized 5-HT agonists to reduce LID in animal 

models (Bezard et al., 2013; Meadows et al., 2018; Stoker & Barker, 2020; Yamada et 

al., 2007) or genetically expression DAT into 5-HT terminals (Sellnow et al., 2019). 

38 

 
Specifically, eltoprazine, a 5-HT agonist, has been demonstrated to be successful in 

preventing LID in a parkinsonian rat model and in non-human primates (Fabbrini & 

Guerra, 2021). These promising results catalyzed a phase I/IIa study in which 

eltoprazine was administered to patients with PD; eltoprazine was successful in  

Figure 1.7: Unregulated Release of DA from a 5-HT Terminal.  
A simplified schematic illustration of the serotonin theory behind LID behavior. Serotonin 
neurons  contain  the  same  enzymes  as  DA  neurons  to  convert  levodopa  to  DA.  Once 
converted, the DA displaces serotonin from their storage vesicles, permitting release of 
DA into the synaptic cleft. However, serotonin neurons do not express DA transporters 
such as DAT for proper  DA reuptake, leading to unregulated DA release and subsequent 
excessive stimulation of DA receptors. Adapted from (Kwon et al., 2022). Abbreviations: 
5-HT = serotonin; AADC = aromatic amino acid decarboxylase; DA = dopamine; DAT = 
dopamine transporter; VMAT2 = vesicular monoamine transporter 2. 

39 

 
 
decreasing LID behavior, although a reduction of levodopa efficacy was also reported, 

contradicting its clinical utility (Bezard, 2013). Ultimately, manipulation of serotonergic 

neurons will only be used if it becomes more efficacious than other drugs such as 

amantadine, which, to-date, is considered the best drug to treat LID (Kwon et al., 2022). 

Amantadine and clozapine (off-label) are currently the only two pharmaceuticals 

known to be efficacious in the treatment of LID (Fox et al., 2018), with the extended-

release amantadine formulation being the only Food and Drug Administration (FDA)-

approved to treat dyskinesias, and marginally most effective, drug (P. Jenner, 2008; 

Konitsiotis et al., 2000; Vijayakumar & Jankovic, 2016). Amantadine, an N-methyl-D-

aspartic acid (NMDA) receptor antagonist, has been shown to stimulate DA release and 

block DA uptake in addition to blocking NMDA receptors. Its administration in a double-

blind, placebo-controlled trial reduced total LID scores by 24% in individuals with low 

level LID without any change to levodopa efficacy (Snow et al., 2000). Unfortunately, 

amantadine is also contraindicated in approximately 25% of patients due to significant 

side effects (see (Hauser et al., 2017)). Because of the mechanism of action of 

amantadine, glutamatergic overactivity has been hypothesized as a mechanism 

responsible for dyskinesia development (Kwon et al., 2022). Similar to other 

pharmaceuticals, amantadine (and clozapine) are not universally effective for all 

patients (Alvir et al., 1993; Postma & Van Tilburg, 1975) and may result in side effect 

development such as ankle edema, hallucinations, and confusion in some patients 

(Fahn, 2003). Fortunately, clinical trials are planned or ongoing to investigate novel 

drugs and treatments to reduce LID behavior (Huot et al., 2022). 

40 

 
Advanced Therapies 

When side effects of pharmacological DRT (e.g., severe LIDs) become 

unmanageable, or when patients have refractory symptoms like dominant tremor, more 

advanced options such as deep brain stimulation (DBS) can be considered. It is 

important to note that DBS, however, is not expected to alleviate levodopa-refractory 

symptoms (e.g., gait freezing) other than tremor. DBS is an invasive procedure that 

involves stereotaxic brain surgery to implant electrodes into the STN and GPi (Follett et 

al., 2010; Grabli et al., 2013; S. K. Kalia et al., 2013; Okun, 2014). The electrodes are 

then connected to a pulse generator placed in the chest (Espay et al., 2018; Fasano et 

al., 2012). The STN and GPi have been approved by the FDA as regional targets for 

DBS, but targeting the GPi has been demonstrated to be superior in reducing LIDs 

compared to the STN (Mansouri et al., 2018). The thalamus is also a brain region that 

has been approved, specifically for tremor-dominant symptoms; however, the thalamus 

is a rarely used DBS target in PD (Bloem 2021). In prospective studies, DBS 

significantly reduced LID behavior, and decreased the patients’ need for medication by 

50-60% (Kleiner‐Fisman et al., 2004). While the mechanism responsible is not well 

understood, it is thought that high frequency stimulation of targeted brain regions (i.e., 

STN and GPi) improve motor function by “normalizing” patterns of neuronal firing (Aum 

& Tierney, 2018; Lozano et al., 2019; Merola et al., 2015).  

To be an eligible candidate to receive DBS, an individual with PD must have a 

good response to levodopa but exhibit severe LIDs and/or medication-resistant tremor, 

or have become refractory to DRT (K. A. Smith et al., 2016). Moreover, several risks of 

DBS exist. Not only are there risks with the surgical procedure itself (e.g., infection, 

41 

 
hemorrhage), or the hardware (e.g., device failure (K. A. Smith et al., 2016; Worth, 

2013), side effects such as cognitive dysfunction and adverse speech development can 

occur (Stoker & Barker, 2020). Also, gait and postural instability symptoms often 

respond poorly (Grabli et al., 2013). DBS is also extremely costly, and patients require 

frequent stimulation adjustments following the procedure (Fahn, 2003). Due to the 

significant side effects and limitations of DBS, clinicians and patients must carefully 

consider DBS as a therapeutic strategy for the parkinsonian symptoms.  

Other advanced treatments are being investigated and implemented as possible 

therapeutic strategies for PD; however, they will not be discussed in-depth here. An 

example of one of these treatments includes the infusion of a levodopa-carbidopa 

intestinal gel which has been FDA approved for almost a decade (Dijk et al., 2020; 

Olanow et al., 2020; Worth, 2013). The goal of the levodopa-carbidopa gel is to achieve 

continuous infusion and reliable absorption of levodopa in order to keep levodopa at 

sufficient levels in the plasma. This procedure, like DBS, is also invasive as it requires 

the patient to undergo an endoscopy to place a gastrostomy tube in the jejunum of the 

large intestine (Espay et al., 2018). As with all other treatment, the benefits and 

drawbacks of advanced therapies must be extensively reviewed by both doctor and 

patient so that the best approach is chosen (Dijk et al., 2020). 

Experimental Disease-Modifying Therapies 

The ultimate goal of treatment development for PD is to generate disease-

modifying therapies that can prevent, delay, or reverse disease progression (Fahn, 

2003; Fox et al., 2018; Noyce et al., 2016; Poortvliet et al., 2020). As was mentioned 

previously, no interventions of this nature currently exist; however, scientists are 

42 

 
continuing to make progress in this area. For instance, gene therapy is a promising 

experimental, potentially disease-modifying, therapy that is being investigated.  

Gene therapy  

Currently, gene therapy has been designed to introduce genes of DA synthesis 

enzymes so that DA can be replenished in the STR of patients with PD (Schuepbach et 

al., 2013). Specifically, the research group of Muramatsu and colleagues (Muramatsu et 

al., 2010) and Christine and colleagues (Christine et al., 2009) utilized gene therapy to 

introduce an adeno-associated virus (AAV) that expressed aromatic amino 

decarboxylase (AADC) into the putamen of PD patients, and patients’ UPDRS scores 

were greatly improved (Christine et al., 2009; Muramatsu et al., 2010). Another example 

is found with lentivirus vector therapy: genes expressing both TH and AADC were 

administered in an open-label phase I clinical trial to patients with PD (OXB-101, (Palfi 

et al., 2014; Stoker & Barker, 2020). Twelve months following treatment, patients 

reported improved UPDRS scores, but not enough to be competitive with other 

treatments. 

A novel gene therapy that has substantial promise to be disease-modifying is 

presented in the studies conducted by Steece-Collier and colleagues (Figure 1.8ab) 

(Caulfield et al., 2025; Caulfield, Vander Werp, et al., 2023; Steece‐Collier et al., 2019). 

Adeno-associated (AAV)-mediated short-hairpin RNA was administered to parkinsonian 

rats to silence striatal voltage-gated, L-type CaV1.3 calcium channels. Delivery of 

CaV1.3 AAV completely prevented LID development, but also strikingly reversed severe 

LID in parkinsonian rats (Figure 1.8c) (Steece‐Collier et al., 2019). Because 

dysregulation of CaV1.3 channels can induce dendritic spine retraction of MSNs (M. Day 

43 

 
et al., 2006; Steece‐Collier et al., 2019), it was theorized that blocking these channels 

could prevent spine retraction, potentially preventing/modifying DA pathophysiology in 

PD. Although promising, there are limitations to gene therapy. Gene therapy is generally 

irreversible, and it can also be difficult to determine/regulate the quantity of gene that is 

delivered (Elkouzi et al., 2019). 

44 

 
a) 

b) 

c) 

Figure 1.8: Silencing of CaV1.3 Channels as a Promising Disease-Modifying Gene 
Therapy for PD.  
Schematic  illustration depicting  (a)  striatal  intraspinous  CaV1.3  channels regulating  the 
influx of Ca2+ into the dendritic spines of MSNs. CaV1.3 channels are normally regulated 
by  D1  and  D2  receptors  (shown  in  yellow)  under  homeostasis.  (b)  However,  when 
degeneration of DA neurons from the SNpc occur in PD, the regulation/inhibition of  

45 

 
 
Figure 1.8 (cont’d) 
CaV1.3  channels through  DA  receptors is diminished,  permitting  an  increased  influx  of 
Ca2+.  This  causes  spine  retraction  and  loss  of  corticostriatal  glutamatergic  inputs.  (c) 
Recent  findings have demonstrated that  inhibition  and/or silencing  of  CaV1.3  channels 
using  an  rAAV-  CaV1.3-shRNA  allow  for  the  maintenance  of  normal  spine  density  on 
MSNs despite severe loss of DAergic neurons, thereby preventing the induction of LID 
(see  Steece-Collier  et  al.,  2019).  Figure  adapted  from  (Caulfield,  Manfredsson,  et  al., 
2023). Abbreviations:  MSN  =  medium  spiny  neurons;  Ca2+  =  calcium  ions;  CaV1.3  = 
voltage-gated L-type calcium channels; DA = dopamine; AAV = adeno-associated virus; 
shRNA = short hairpin ribonucleic acid. For more details on these findings, see (Caulfield 
et al., 2025; Caulfield, Vander Werp, et al., 2023). 

Targeting α-synuclein pathology 

Along with targeting DA degeneration, other potentially disease-modifying 

experimental therapies also target α-synuclein pathology. Gene therapy has also been 

utilized for this: gene-silencing mechanisms that target messenger RNA of α-synuclein 

has been attempted to reduce the synthesis of α-synuclein (Fields et al., 2019; Savitt & 

Jankovic, 2019). Immune therapy is also of interest. Specifically, in Phase I clinical trials, 

a humanized monoclonal antibody that targets aggregated α-synuclein (prasinezumab) 

resulted in a 97% reduction in free serum α-synuclein (Jankovic et al., 2018; Schenk et 

al., 2017). Because of the success of the Phase I clinical trial, a Phase II clinical trial is 

now ongoing (NCT03100149).  

Interestingly, several drugs on the market are being repurposed because of their 

ability to reduce α-synuclein pathology. The glucagon-like peptide 1 (GLP-1) analogue, 

exenatide, is a fitting example. GLP-1 has historically been used to treat type-2 

diabetes; however, individuals with PD have exhibited improved cognitive and motor 

function following GLP-1 treatment (Stoker & Barker, 2020). Cell and animal models of 

nigral degeneration have also demonstrated a neuroprotective effect in response to 

GLP-1 administration (Bertilsson et al., 2008; Harkavyi et al., 2008; Y. Li et al., 2009). 

Another drug, terazosin, which is an α1-adrenergic antagonist usually used to treat 

46 

 
benign prostatic hypertrophy, has shown a reduction in α-synuclein in transgenic mice 

and in neurons from patients with LRRK2 mutations (Cai et al., 2019). Despite the 

promise of these studies, the entirety of physiological functions of α-synuclein remains 

to be determined, and thus, it is therefore important to keep in mind that there may be 

negative consequences of decreasing endogenous α-synuclein too much (Collier et al., 

2016; Elkouzi et al., 2019; Gorbatyuk et al., 2010; Stoker & Barker, 2020).  

As discussed previously, it is well established that, by the time classic motor 

symptoms manifest in PD patients, a significant loss of SNpc DA neurons has already 

occurred (Fearnley & Lees, 1991; Noyce et al., 2016). Therefore, although DRTs (i.e., 

levodopa) and other therapies are successful at treating motor symptoms, there has yet 

to be developed a therapy that can prevent or reverse the pathology of PD. Pathways 

that have been experimentally implicated in PD (e.g., lysosomal and mitochondrial 

dysfunction, neuroinflammation) are currently being investigated as possible drug 

targets, with the goal of treating PD prior to motor symptom development (Jankovic, 

2008; K. S. P. M. P. Jenner & Olanow, 2007; Pan et al., 2008). A promising experimental 

therapy aimed at re-establishing the nigrostriatal DA system that remains of worldwide 

interest is cell transplantation therapy. Understanding factors that impact the benefits 

and limitations of cell transplantation is the major focus of my dissertation research. 

47 

 
 
 
Regenerative Cell Transplantation Therapy 

Brief History of Cell Transplantation  

The concept of neural transplantation into the adult mammalian brain has been of 

interest for almost four centuries, but it was not until 1890 that the first experimental 

attempt at transplantation was successfully conducted. W. Gilman Thompson, an 

American physician, was the first to attempt transplantation. Briefly, in one of his 

studies, cortical tissue from the occipital lobe of dogs was excised and subsequently 

transplanted into the occipital lobe of recipient dogs or cats. Remarkably, when 

examined histologically after seven weeks, there seemed to be survival of the donor 

tissue, with a mix of healthy and degenerating cells (Dunnett, 2009; Thompson, 1890a, 

1890b). While this seemed to be a promising finding, the methods of the time were 

limited. Therefore, it was more likely that the transplanted tissue had died and left scar 

tissue or host-derived immune cells in its place (Bjorklund & Stenevi, 1985; Dunnett, 

2009). Despite how profound these findings were at the time, Thompson’s studies 

unfortunately elicited little immediate follow-up experimentation.  

Almost twenty years later, in 1907, another attempt was made to demonstrate 

that grafting into the adult mammalian brain was indeed possible. This was performed 

by Del Conte who grafted non-neuronal embryonic tissue into the cerebral cortex of 

adult dogs. Similar to Thompson’s work, Del Conte demonstrated partial tissue survival; 

however, he believed survival to be only temporary (Bjorklund & Stenevi, 1985; Del 

Conte, 1907). Following these experiments, several grafting studies involving peripheral 

nerve transplantation or other non-central nervous system (CNS) tissue transplant 

studies were conducted spanning across the next decade.  

48 

 
Finally, in 1917, Elizabeth Dunn successfully demonstrated that cortical tissue 

transplanted between neonatal rat pups could survive, albeit with a poor survival rate at 

less than 10% (Bjorklund & Stenevi, 1985; Dunn, 1917). Despite a minute survival rate, 

Dunn was credited with the first successful evidence that CNS tissue could survive, at 

least to some extent, in the brain (Dunnett, 2009). With these findings, Wilfrid Le Gros 

Clark went on to provide evidence of survival of embryonic cortical tissue into the 

neonatal brain of immature six-week-old rabbits. He discovered that these cells could 

not only survive but also differentiate into mature neurons in the host neocortex (W. E. 

L. G. Clark, 1940; Dunnett, 2009).  

Throughout the next few decades, the scientific community still remained 

skeptical of the ability of transplanted neurons to fully integrate and differentiate into the 

adult mammalian brain. Then, in the early 70s, Gopal Das and Joseph Altman launched 

what is now considered the “modern era” of neural transplantation (Bjorklund 1999, 

Dunnet 2009). In their research, they injected [3H] thymidine into the cerebellar cortex of 

neonatal rat pups to label still-proliferating cells. Their results demonstrated that the 

labeled cells successfully survived, migrated, and differentiated into proper neuronal 

phenotypes when engrafted into the host cerebella (Das & Altman, 1971; Dunnett, 

2009). Following Das’ and Altman’s work, additional research teams endeavored to 

further study the intricacies of cell transplantation. Using new anatomical techniques for 

the time, groups including Olson and Seiger (Dunnett, 2009; Olson & Seiger, 1972) and 

a Swedish group at the University of Lund led by Anders Bjorklund (Björklund & Stenevi, 

1971; Stenevi et al., 1976) collected evidence that allowed scientists to determine 

optimal development ages for survival, differentiation, and growth of grafted tissue in the 

49 

 
host brain. A comprehensive exploration of the full history of cell transplantation can be 

found in (Bjorklund & Stenevi, 1985; Dunnett, 2009).  

Preclinical and Early Clinical Trials of Cell Transplantation in PD 

In 1979, two independent groups published promising evidence of the functional 

benefit of SN grafts specifically in a parkinsonian rat model (Bjorklund & Stenevi, 1979; 

Perlow et al., 1979). Following unilateral lesions of the nigrostriatal DA pathway (6-

hydroxydopamine (6-OHDA) injections), Bjorklund and colleagues transplanted 

embryonic ventral mesencephalic (eVM) tissue containing the developing SN DA 

neurons into a cerebral cortical cavity overlying the STR (Bjorklund & Stenevi, 1979). 

Simultaneously, Perlow and colleagues dispersed eVM tissue into the lateral ventricles 

(Perlow et al., 1979). In both cases, these grafts, which contained the developing DA 

neurons, reduced rotational asymmetry compared to non-grafted lesioned rats, 

suggesting a restoration of motor deficit. While nigrostriatal synaptic connectivity was 

seemingly restored in the study conducted by Bjorklund and colleagues, results from 

Perlow and colleagues indicated that diffusion of DA from the ventricle was the reason 

for behavioral improvement, not successful graft-host connectivity (Björklund & Lindvall, 

2017b).  

Soon after these two experiments, a transplantation technique of stereotaxically 

inserting eVM neurons directly into the STR (Bjorklund et al., 1980; Björklund et al., 

1983; R. H. Schmidt et al., 1981) was developed and proven to achieve widespread 

reinnervation. Numerous preclinical studies have since been conducted in order to 

optimize functional outcomes, addressing issues related to experimental protocols of 

cell transplantation including cell preparation and source, graft delivery method, 

50 

 
immunological responses, and cell storage (Dunnett, 2009; Freeman & Widner, 1998). 

Although other various cell sources and transplant locations have been studied, the 

most promising approach to-date has been transplanting eVM DA neurons directly into 

the STR (Steece-Collier & Collier, 2016). Due to promising evidence of preclinical trials, 

interest in clinical application of cell transplantation in PD increased rapidly.  

The first clinical trials of neural transplantation in individuals with PD occurred in 

1982 and 1983, respectively. Two patients received implantations of their own adrenal 

medulla cells, which secrete catecholamines including DA, into the caudate nucleus. 

However, only transient improvement of motor function occurred (Backlund et al., 1985; 

Björklund & Lindvall, 2017a; Lindvall et al., 1987). In another clinical trial conducted by a 

group in Mexico City, two young patients with PD (35 and 39-years-old) also received 

adrenal medullary autografts to the caudate nucleus. Results from this study 

demonstrated a significant reduction in rigidity, tremor, and akinesia in these patients 10 

months following transplantation (Madrazo et al., 1987). Unfortunately, a larger clinical 

trial involving 61 patients with PD from the US and Canada who were recipients of 

adrenal medullary grafts, could not replicate the results from the Mexico City trial: few 

patients (19%) showed improvement 2 years after surgery, and morbidity/mortality was 

relatively high in a sizable portion of these patients (Goetz et al., 1991).  

51 

 
 
Table 1.1: Current Planned or Ongoing Clinical Trials for Cell Transplantation in PD. 
Abbreviations: iPSCs = induced pluripotent stem cells; PASCs = pluripotent stem cells  

52 

Trial ID Location Cell Source Enrollment Phase Status NCT06687837 Boston, MA, USA Autologous iPSCs 8 Phase I Recruiting NCT06482268 La Jolla, CA, USA Human iPSCs 7 Phase I Recruiting NCT06422208 Boston, MA, USA Autologous iPSC-derived DA neurons 6 Phase I Enrolling my invitation NCT06141317 San Jose, Costa Rica PASCs 40 Phase I/II Active, not recruiting NCT05901818 Beijing, China Autologous induced neural stem cell-derived DA precursor cells 10 Phase I Recruiting NCT05699161 Leon, Nicaragua Adipose-derived stromal vascular fraction cells 10 Phase I/II Completed NCT05691114 Shanghai, China hAESCs 18 Phase I Recruiting NCT05635409 Lund, Sweden (STEM-PD) hESCs 8 Phase I Active, not recruiting NCT05435755 Shanghai, China hAESCs 12 Early Phase I Unknown status NCT05094011 Unknown Adipose-derived mesenchymal stem cells 9 Phase I Not yet recruiting NCT04414813 Shanghai, China hAESCs 3 Early Phase I Completed NCT04146519 Minsk, Belarus Autologous mesenchymal stem cells 50 Phase II/III Unknown status  
 
Table 1.1 (cont’d)  
isolated from adipose tissue; DA = dopamine; hAESCs = human amniotic epithelial stem 
cells;  hESCs  =  human  embryonic  stem  cells;  MSCs  =  mesenchymal  stem  cells;  ISC-
hpNSC = International Stem Cell Corporation human parthenogenetic neural stem cells 

53 

Trial ID Location Cell Source Enrollment Phase Status NCT03119636 Zhengzhou, Henan, China hESCs 50 Phase I/II Unknown status NCT02780895 Mexico City, Mexico hFSCs 8 Phase I Unknown status NCT02611167 Houston, TX, USA Allogenic bone marrow-derived MSCs 20 Phase I Completed NCT01860794 Seongnam-si, Gyeonggi-do, Korea Fetal mesencephalic neuronal precursor cells 15 Phase I/II Unknown status NCT01446614 Guangzhou, Guangdong, China Autologous bone marrow-derived mesenchymal stem cells 20   Phase I/II Unknown status NCT00226460 Tampa, FL, USA (Neurocell-PD) Fetal porcine cells Unknown Phase II Completed NCT02452723 Melbourne, Victoria, Australia ISC-hpNSC 12 Phase I Unknown status NCT01898390 Unknown (TRANSEURO) Allografts of fetal ventral mesencephalic tissue 13 N/a, open label Completed JMA-IIA00384 Kyoto, Japan Allogenic human iPSCs 7 Phase I/II Completed   
 
 
Following these initial clinical trials, scientists began to shift their focus to utilizing 

human embryonic neuronal tissue instead of transplanting adrenal medullary cells. In 

the late 1980s, a group at the University of Lund conducted preclinical transplantation 

trials of human eVM tissue engrafted into immune-suppressed parkinsonian rats 

(Björklund & Lindvall, 2017a; Brundin et al., 1986, 1988; Clarke et al., 1988). These DA 

neurons were shown to successfully survive and reinnervate the STR, providing notable 

functional benefit to parkinsonian animals. Due to these incredible results in a preclinical 

rat model, two patients at Lund finally underwent transplantation of eVM neurons into 

the caudate and putamen in 1989. Remarkably, patients in this open-label trial exhibited 

successful survival of their grafted neurons, and the grafts rescued both spontaneous 

and drug-induced DA release (Björklund & Lindvall, 2017b; Lindvall et al., 1990). After a 

one-year follow-up, patients still exhibited clinical benefit with improved OFF-time motor 

function (Lindvall et al., 1992). Additional clinical trials confirmed these results, 

demonstrating that, collectively, individuals with PD who received primary DA neuron 

transplants can exhibit remarkable functional improvement (Cochen et al., 2003; 

Freeman et al., 1995; Kordower et al., 1998; Lindvall & Hagell, 2000; Mendez et al., 

2002; Peschanski et al., 1994; Piccini et al., 1999).  

These notable findings encouraged the National Institute of Health (NIH) to fund 

two double-blind, placebo-controlled trials of cell transplantation in the mid-1990s in the 

US (Freed et al., 2001; Olanow et al., 2003). Results from these trials, when 

categorized by patient age and disease severity, demonstrated that primary DA neuron 

grafts can survive, function, and restore DA release in the putamen of PD patients 

(Freed et al., 2001; Olanow et al., 2003). Despite promising results, however, there was 

54 

 
an unfortunate occurrence of novel OFF-medication behaviors known as graft-induced 

dyskinesia (GID) in 56.5% of the study participants in (Olanow et al., 2003). These GID 

behaviors developed 6-12 months following the transplantation procedure after 

cessation of immunosuppression. The manifestation of GID in these and other trials 

regrettably summoned a worldwide moratorium on all clinical grafting trials for PD 

(Hagell et al., 2002a). Until the underlying mechanisms, and subsequent prevention, of 

GID can be elucidated and achieved, grafting cannot be considered a fully optimized 

option for PD treatment (see Table 1.1 for currently ongoing/planned clinical trials).  

The Unanticipated Side Effect of Cell Transplantation: Graft-Induced Dyskinesia (GID)  

Graft-induced dyskinesias (GID) are defined as abnormal involuntary OFF-

medication behaviors that develop only in individuals who received primary neural 

transplants (for review (Maries et al., 2006; Steece-Collier et al., 2012)). These GID 

profiles develop as the graft matures and as the typical pre-graft LID behaviors 

disappear (Steece-Collier et al., 2012) both in humans and in animal models (Lane et 

al., 2006; Maries et al., 2006; Soderstrom et al., 2008). Clinically, GIDs tend to manifest 

as more focal stereotypic movements in contrast to that of LID (Freed et al., 2001), 

often localized to either the upper or lower extremities correlating with graft placement 

(Hagell et al., 2002a; Maries et al., 2006; Olanow et al., 2003). Moreover, GIDs bear 

resemblance to diphasic drug-induced dyskinesia (Hagell & Cenci, 2005); however, 

unlike LIDs, GIDs cannot be alleviated by lowering the dose of levodopa. In the clinical 

trials discussed above, GID severity varied from mild for some patients, to severe, in 

which some had to undergo STN DBS to reverse the aberrant effects of their grafts 

(Freed et al., 2001). While several underlying mechanisms of GID have been 

55 

 
postulated, this remains a topic of controversy. These mechanisms include, but are not 

limited to, pre-graft levodopa history, age of recipient, donor cell source, presence of 

non-DA neurons (e.g., serotonergic neurons), uneven DA reinnervation/excess DA 

release, host-immune response, and/or asymmetric synaptic connections between the 

host and donor (for review (Steece-Collier et al., 2012)). Although not to an exhaustive 

level, some notable proposed mechanisms of GID are discussed below.  

Modeling Graft-Induced Dyskinesia  

In preclinical laboratories, rodent models are utilized to experimentally study GID 

behavior. Commonly, parkinsonism will first be induced in rodent models via unilateral, 

intranigral 6-OHDA injections in order to lesion the nigrostriatal pathway. Embryonic VM 

graft tissue will then be transplanted into the parkinsonian striatum. Experimental GID in 

these animals are then induced with either levodopa or amphetamine administration 

(Figure 1.9).  

Following administration of levodopa, grafted rodents will develop focal, 

stereotypic, and repetitive movements similar to what is seen in human subjects (Hagell 

& Cenci, 2005; Maries et al., 2006; Soderstrom et al., 2008). Affected bodily regions are 

also comparable to GID expression, specifically in individuals from the Denver/Columbia 

clinical trial (Freed et al., 2001). In response to amphetamine administration, GID 

resemble a more robust, widespread dyskinetic profile similar to LID; however, they are 

only observed in the presence of a DA graft and as the graft matures. In our laboratory, 

we have more recently relied on amphetamine administration to induce GID based on 

the finding that DA-grafted, and not sham-grafted, rats demonstrate robust dyskinetic 

behavior in response to low-dose amphetamine (Lane, Brundin, et al., 2009b; Lane, 

56 

 
Vercammen, et al., 2009; Shin et al., 2012b; G. A. Smith, Breger, et al., 2012; G. A. 

Smith, Heuer, et al., 2012). 

Figure 1.9: Modeling Experimental GID in Rodents.  
Schematic diagram illustrating that plasma DA levels must be elevated with amphetamine 
(or levodopa) administration in rats to “push” the animal into a diphasic-like dyskinesia 
range, phenotypically like the GID behavior seen in engrafted patients with PD. Upon low-
dose  amphetamine  administration,  grafted  parkinsonian  rats  will  develop  focal, 
stereotypic  movements  that  characterize  GID  comparable  to  grafted  human  patients. 
Adapted  from  (Steece-Collier  et  al.,  2012).  Abbreviations:  LD  =  levodopa;  amph  = 
amphetamine.  

While spontaneous, non-medicated GIDs can occur in rodent models, they occur 

sporadically, and in their active phase (i.e., dark), making behavioral evaluation almost 

impossible. The phenomenon of requiring pharmacological agents to raise plasma DA 

levels in animal models arguably remains the only major discrepancy between 

experimental preclinical studies and clinical human trials of GID. Regardless, the 

appearance of GID, both in humans and in rodents, only manifests after grafting as the 

cells mature and is not seen preoperatively (T. Carlsson et al., 2006; Lane et al., 2006; 

57 

 
 
 
Lane, Vercammen, et al., 2009; Maries et al., 2006; Soderstrom et al., 2008). Further 

limitations of neural grafting will be discussed later on.  

Postulated Mechanisms Underlying GID Behavior  

There remains contention in the field of neural transplantation as the underlying 

mechanisms responsible for GID behavior have not yet been elucidated. While some 

are confident that the presence of non-dopaminergic neurons in the grafts are the culprit 

(see below), preclinical and clinical evidence suggests against this notion. Some have 

shown that the size of the graft itself impacts GID: grafted parkinsonian rats with large 

grafts demonstrated increased GID severity compared to smaller grafts (Lane et al., 

2006). In contrast, another study demonstrated that focal, not widespread, grafts induce 

GID behavior in parkinsonian rats (Maries et al., 2006). Other groups have also 

revealed that the degree of disease severity or the severity of preoperative LID behavior 

correlates the development of GID (García et al., 2011; Lane, Brundin, et al., 2009b; 

Rylander Ottosson & Lane, 2016; Tronci et al., 2015). Additional components that have 

been considered include immune response (Soderstrom et al., 2008), age of the graft 

recipient, and preoperative cell storage. Consequently, clinical researchers have 

endeavored to modify and optimize factors such as patient selection and cell 

composition prior to transplantation; however, the mystery of GID remains. In the 

following section, five prominent postulated GID mechanisms relating to my studies are 

discussed: the presence of non-DA neurons, the immune response, abnormal graft-host 

synaptic circuitry, uneven dopamine innervation/excessive DA release, and 

DA/glutamate co-transmission.  

58 

 
Presence of Non-DA Neurons/Cellular Components 

The cellular composition of eVM grafts, specifically the presence of 5-HT 

neurons, has been suggested as a possible underlying factor responsible for GID 

behavior. While the 5-HT system in the DA-denervated brain (i.e., PD) has been linked 

to LID following administration of levodopa (Carta et al., 2007; Lindgren et al., 2010; 

Rylander et al., 2010; Sellnow et al., 2019; H. Tanaka et al., 1999), there remains a lack 

of consensus on the role of this system in the development of GID. The hypothesis of 5-

HT and GID is largely based on the biological ability of 5-HT neurons to convert, store, 

and release DA due to having similar cellular machinery. For instance, it is well-known 

that 5-HT neurons can take up exogenous levodopa, convert it into DA, and store DA in 

its vesicles via the vesicular monoamine transporter 2 (VMAT2) found in both DA and 5-

HT neurons (Tronci et al., 2015). However, because 5-HT neurons do not possess 

dopamine transporters (DAT) for DA reuptake, and do not have regulatory DA 

autoreceptors on their terminals, DA continues to be released and left in the synaptic 

cleft, theoretically leading to GID behavior (Politis, 2010).  

Many preclinical rodent model studies have collected evidence in favor of this 

hypothesis, demonstrating the presence of 5-HT+ neurons within intrastriatal eVM 

transplants (Winkler et al., 2005). Using bimodal chemogenetic (DREADD) activation of 

5-HT receptors, Aldrin-Kirk and colleagues observed substantial GID induction in 6-

OHDA-lesioned rats (Aldrin-Kirk et al., 2016). Likewise, in grafted patients with PD, two 

individuals who exhibited significant GID behavior developed excessive 5-HT 

innervation from their grafts. Following administration of buspirone, which is a partial 5-

HT1a agonist, GID behavior in these patients was attenuated (Politis et al., 2010; Shin 

59 

 
et al., 2012a). In another study also conducted by Politis, positron emission topography 

(PET) and single photon emission computed tomography (SPECT) imaging revealed an 

elevated 5-HT/DA ratio within eVM neurons that were transplanted into an individual 

with PD (Politis et al., 2011). It is important to note that, in another study where grafted 

patients had abundant 5-HT neurons in their grafts, they did not develop GID (Mendez 

et al., 2008).  

To contrast the evidence in favor of the role of the 5-HT system in GID, other 

research has shown that GID can develop in the absence of 5-HT neurons. For 

instance, histological results collected by Lane and colleagues of 6-OHDA-lesioned rats 

following intrastriatal VM transplantation revealed very low numbers of 5-HT+ neurons 

despite GID expression (Lane, Brundin, et al., 2009a). Further, the 5-HT/DA cell ratio 

within grafted VM grafts was not shown to be significantly correlated with GID behavior 

in parkinsonian rat models (García et al., 2012; Mercado et al., 2021). Lastly, an 

experiment that transplanted DA-only, DA + 5-HT, or 5-HT-only grafted neurons into 6-

OHDA-lesioned rats demonstrated that only the recipients of either DA-only or DA + 5-

HT neurons exhibited amphetamine-mediated GIDs. 5-HT alone did not induce aberrant 

behavior (Shin et al., 2012b), suggesting that the DA system within grafted neurons may 

provide more of a contribution to GIDs (Aldrin-Kirk et al., 2016; García et al., 2012; 

Lane, Brundin, et al., 2009b; Rylander Ottosson & Lane, 2016).  

A reasonable, biological explanation for the divergency of the above studies is 

that 5-HT neurons may, instead, play more of a modulatory, instead of a direct, role in 

GID development. When given concomitantly with a DAT blocker, fluvoxamine (i.e., 

serotonin transporter (SERT) blocker) administration significantly increased GID 

60 

 
expression in 6-OHDA-lesioned parkinsonian rats (Lane et al., 2006). Another 

experiment similarly co-administered 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-

DPAT; a 5-HT agonist) with raclopride (D2 receptor antagonist) to parkinsonian rodents, 

and this co-administration suppressed amphetamine-mediated axial and limb GIDs 

(Lane, Brundin, et al., 2009b). Moreover, eticlopride administration alone, also a D2 

antagonist, suppressed GIDs in parkinsonian rats (Shin et al., 2012a). Lastly, while 

buspirone is a 5-HT receptor partial agonist, it also displays DA D2 antagonism (for 

review (Steece-Collier et al., 2012)). Collectively, the evidence seemingly points to more 

of a key role of the dopaminergic system in GID behavior (discussed further in the 

“Uneven DA reinnervation/DA release” section). While the 5-HT-GID hypothesis remains 

controversial as patients still express GID behavior with low 5-HT expression, clinical 

trials have since attempted to minimize the inclusion of 5-HT neurons prior to 

transplantation to lower the potential risk of GID exhibition in patients with PD (Lane & 

Lelos, 2022).  

Immune Response 

The host immune response has been a major area of debate in the field of neural 

transplantation (Tronci et al., 2015). Historically, the CNS was considered to be an 

immuno-privileged site; however, moderate immune activation does occur in the brain 

following ectopic cell engraftment, mostly due to the necessity of having to use non-

genetically identical allografts in human subjects (for review (Steece-Collier et al., 

2012)). Several clinical trials have reported the presence of immune markers such as 

activated microglia surrounding the grafted cells in immunohistochemical postmortem 

analyses of grafted patients with PD (Freed et al., 2001; Kordower et al., 1997; Olanow 

61 

 
et al., 2003; Winkler et al., 2005). Significantly elevated levels of activated microglia and 

astrocytes surrounding DA grafts transplanted into parkinsonian rat models has also 

been reported, sharply contrasting a lack of microglia and astrocytes in non-DA control 

grafts (Lane & Lelos, 2022; Soderstrom et al., 2008).  

Not only have these studies marked the presence of microglia and astrocytes 

surrounding grafted DA neurons, research has also pointed to a probable role of the 

immune response in the induction of GID behavior clinically. For instance, in the Tampa-

Mount Sinai trial in which low-dose immunosuppressive medication was given for six 

months following grafting surgery, patients developed GID behavior only after 

immunosuppression was ceased (Olanow et al., 2003). The Denver/Columbia trial, 

which did not offer any immunosuppression, similarly reported GID behavior in grafted 

patients with PD (Freed et al., 2001). Likewise, in our primary DA-grafted parkinsonian 

rat model, GID behavior emerged, and increased, following exposure to immune 

activation via injections of peripheral spleen cells (Soderstrom et al., 2008). Curiously, 

GID did not manifest in grafted parkinsonian rats who received tissue from the same 

inbred strain (i.e., syngeneic grafts), further confirming a role of the host-immune 

response in GID development (Soderstrom et al., 2008; Steece-Collier et al., 2012).  

Mechanistically, immune activation has been proposed to cause GID by a few 

mechanisms. First, immune activation has potential to cause diminished graft cell 

survival, ultimately affecting the ability of the graft to effectively integrate into the host 

and successfully restore DA levels in the STR (Hagell & Cenci, 2005; Hudson et al., 

1994; Tronci et al., 2015). Another possibility is the release of pro-inflammatory 

cytokines that could activate specific signaling pathways or remodel synaptic 

62 

 
connections, both of which could lead to the development of dyskinetic behavior (for 

review (Hagell & Cenci, 2005)). Indeed, cytokines released from inflammatory immune 

cells activated nuclear signaling pathways that increased Fos protein expression in 

striatal neurons, which was correlated with the development of LID in animal models 

(Andersson et al., 1999; Hagell et al., 2002a; Winkler et al., 2002). Moreover, DA is 

known to have a modulatory effect on astrocytes and microglia, both of which express 

D1- and D2-like receptors (Boyson et al., 1986; Färber et al., 2005; Miyazaki et al., 

2004). In this way, transplanting exogenous DA-producing cells would be expected to 

induce immune cell infiltration, activation, and cytokine release from astrocytes and 

microglia in the host (Lane & Lelos, 2022).  

As more research is conducted on the connection between the host-immune 

response and GID induction, understanding whether immunosuppressive therapies in 

preclinical animal models eliminates GID may be an important next-step in elucidating 

its underlying mechanisms. It would also be important to reveal which specific immune 

components (e.g., microglia, complement factors) are seemingly permissive to these 

aberrant GID behaviors. More specifically, some immune factors could affect synapse 

formation between the grafted DA neurons and the host MSNs, potentially leading to 

GID (for review (Steece-Collier et al., 2012)). This could offer an explanation as to why 

there is an increase in the percentage of atypical, asymmetric synapses formed by 

engrafted DA neurons in GID+ patients with PD and grafted parkinsonian rats (see the 

“Abnormal Graft-Host Synaptic Circuitry” section). In Chapter 3 and 4, correlations 

between well-known immune markers and GID expression is experimentally 

investigated in our DA-grafted parkinsonian rat model.  

63 

 
Abnormal Graft-Host Synaptic Circuitry  

Another hypothesis theorized to be responsible for GID behavior includes 

abnormal graft-host synaptic circuitry. Grafted eVM neurons establish synaptic 

connections with host striatal MSNs (Bolam et al., 1987; Kordower et al., 1996); 

however, it is more than possible that the grafted neurons fail to restore proper synaptic 

circuitry onto host MSNs, leading to signaling abnormalities that could potentially 

underlie GID (Hagell & Cenci, 2005). While DA neurons normally form en passant (i.e., 

in passing), symmetrical appositions, largely devoid of defined synaptic characteristics, 

onto the dendritic spines of MSNs (Gerfen & Surmeier, 2011; W. Shen et al., 2016), 

increasing evidence indicates that DA neurons in grafted subjects exhibit abnormal, 

atypical asymmetric synaptic connections (i.e., axodendritic or axosomatic) onto host 

neurons. The functional asymmetric synaptic connections are characteristic of excitatory 

neurotransmission (e.g., glutamatergic transmission) (Peters & Palay, 1996). In this way, 

it is reasonable to suggest that an increase in the formation of asymmetric synapses 

between grafted DA and host neurons could lead to the development of dyskinesia (i.e., 

GID) (Morgante et al., 2006; Picconi et al., 2003). 

Various research groups have collected evidence in favor of the abnormal 

synaptic circuitry hypothesis. For instance, using ultrastructural and 

immunohistochemical analysis, Freund and colleagues and Mahalik and colleagues 

demonstrated that striatal DA grafts formed aberrant connections with host MSNs in a 6-

OHDA parkinsonian rat model, specifically onto host cell bodies (T. Freund et al., 1985; 

Mahalik et al., 1985). Arguably the most promising findings were demonstrated by the 

Steece-Collier group in 2008. In a DA-grafted parkinsonian rat model, our group 

64 

 
demonstrated, ultrastructurally, that the grafted DA neurons made asymmetric synapses 

directly onto the host dendrites or the cell somas, and this was strongly correlated with 

the exhibition of GID behavior in these animals (Soderstrom et al., 2008). Interestingly, 

this phenomenon has also been importantly noted in human postmortem tissue from 

grafted patients with PD; asymmetric connections made by grafted DA neurons were 

also observed ultrastructurally (Kordower et al., 1997).  

Not only have asymmetric synapses been detected in preclinical rodent models 

and in patients with PD, atypical synapses have been observed in non-human primate 

parkinsonian models as well. In MPTP-lesioned primates, 67% of transplanted DA 

neurons exhibited axodendritic connections, 32% axosomatic connections, and only 

1.33% onto dendritic spines (Leranth et al., 1998). This is in comparison to the control 

primates that had 97% of DA terminals that terminated onto the host MSN spines (i.e., 

normal symmetric associations). Despite the evidence of abnormal graft-host circuitry in 

this non-human primate model of neural grafting, it is important to mention that GIDs 

have never been observed in primates, even after levodopa or amphetamine treatment, 

so the phenomenon in this model cannot be correlated, yet, to GID behavior (Kordower, 

Vinuela, et al., 2017). Nevertheless, the abundance of evidence collected thus far 

deems in favor of the abnormal graft-host synaptic connectivity hypothesis underlying 

GID, and if correct, enhancement of physiological synapse formation between grafted 

DA and host neurons could effectively ameliorate GID in both patients and animal 

models of PD.  

65 

 
Uneven DA reinnervation/DA release 

Preclinical animal models and clinical grafting trials have pointed to the possibility 

of uneven DA reinnervation and/or excess DA release in association with GID 

development following engraftment of eVM DA neurons. However, study results remain 

indefinite or contradictory. In grafted individuals with PD, clinicians have performed 18F-

DOPA (fluorodopa; FD) PET scans in order to directly measure DA storage capacity and 

indirectly assess DA innervation (Hagell & Cenci, 2005). With these scans, researchers 

have demonstrated that VM grafts can normalize FD uptake in the grafted striatum 

(Piccini et al., 1999). Further, FD values of grafted patients were found to be 

significantly increased in patients who also developed GID compared to those who did 

not (Ma et al., 2002). Remarkably, in the Denver/Columbia clinical grafting trial, patients 

who expressed GID behavior had twice the amount of FD PET signals compared to 

patients who did not develop GID at 12 months following transplantation; at 24 month 

post-transplantation, levels were almost three times larger (Ma et al., 2002).  

Despite demonstrable promise for the role of excess DA release, other clinical 

trials have failed to provide comparable results. For instance, in a retrospective analysis 

conducted by Hagell and colleagues, GID scores were not found to be correlated with 

postoperative FD uptake (Hagell et al., 2002b). Similarly, Olanow and colleagues 

reported a lack of correlation between GID and FD uptake in the putamen (Olanow et 

al., 2003). Not only were they not able to find a correlation in patients, preclinical animal 

studies have likewise demonstrated a lack of association between GID and FD (Cragg 

et al., 2000; Doucet et al., 1990; Kirik et al., 2001). A key issue of these collective 

clinical trials and preclinical animal studies is, even if an increase in FD uptake was 

66 

 
demonstrated (Ma et al., 2002), DA uptake failed to exceed supranormal DA levels or 

innervation of the intact STR (Hagell et al., 2002a; Ma et al., 2002; Olanow et al., 2003), 

arguing against the theory of excess DA release, or at least widespread excess release. 

Because of this, Hagell et al., 2002 has posited that OFF-medication dyskinesia (i.e., 

GID) do not result from excessive innervation of grafted DA neurons (Hagell et al., 

2002b).  

Although the postulation from Hagell and colleagues is not in favor of excessive 

DA release from the grafts, the possibility of this phenomenon underlying GID behavior 

should not be completely denied. An alternative explanation that has been offered is 

that GIDs result from of so-called “hotspots” of DA activity due to uneven patterns of DA 

release and/or reinnervation. Indeed, evidence from Ma Y and colleagues showed that 

FD uptake was increased in GID+ patients but signals were localized to only two zones 

within the left putamen (Ma et al., 2002). Additional evidence has established that more 

focal VM grafts, either transplanted at two separate striatal sites (Lane et al., 2006) or at 

a single “hotspot” site (Maries et al., 2006), induced GID behavior in parkinsonian rats. 

In contrast, VM tissue transplanted and distributed at six sites, which provided more 

widespread graft-derived reinnervation, significantly reduced GID induction (Maries et 

al., 2006). In this way, imbalanced DA reinnervation may be a more appropriate 

pathogenic theory potentially underlying GIDs.  

While current clinical trials and preclinical animal studies exhibit contrarian 

evidence, it is imperative to note the limitations of these studies. Most importantly, FD 

PET uptake widely used in the above studies does not directly show DA release. It only 

measures the capacity of the grafted neurons to uptake and synthesize DA. 

67 

 
Undoubtedly, this technique is a valuable tool; however, it does not illustrate the 

intricacies of DA signaling/release occurring in grafted VM neurons. Certainly, in spite of 

the lack of evidence for VM grafts releasing excess DA, other clinical trials have 

revealed that buspirone administration (a DA D2 receptor antagonist) successfully 

reduced GID severity in grafted patients (Politis et al., 2010, 2011; Steece-Collier et al., 

2012), yet another piece of evidence in favor of a role for DA release. Therefore, the 

connection between DA release and/or uneven DA innervation and GID behavior should 

continue to be investigated in clinical research until fully elucidated.  

DA/glutamate co-transmission 

Due to the complex nature of GID behavior, it is more than likely that one 

mechanism alone does not solely cause GID. For example, excessive DA release/DA 

reinnervation alone may not cause GID, but in combination with abnormal synaptic 

circuitry, it could underlie GID. Of relevance, DA neurons have been shown to have the 

potential to co-release DA and other neurotransmitters, including glutamate. While this 

has been known for some time, the functional significance of dual neurotransmission 

remains unclear. Many research groups have reported that a subpopulation of DA 

neurons can co-express both DA and glutamate, evidenced by co-expression of 

vesicular glutamate transporter 2 (VGLUT2) in tyrosine hydroxylase-positive (TH+) 

neurons (Bérubé‐Carrière et al., 2009; Buck et al., 2022; Dal Bo et al., 2004; Fortin et 

al., 2019; Mercado et al., 2021, 2024; Mingote et al., 2019; Root et al., 2016; H. Shen et 

al., 2018; Sulzer et al., 1998; Trudeau et al., 2014). Confirmed behaviorally, reduced 

locomotion in mice following cocaine administration (Hnasko et al., 2010) and 

methamphetamine (H. Shen et al., 2021) was reported in knock-out mice of VGLUT2 

68 

 
expression in DA neurons. Hnasko and colleagues also demonstrated, in slice culture of 

VGLUT2 knock-out DA neurons, that glutamate and DA release was significantly 

decreased, further supporting an important function of DA/glutamate co-transmission 

(Hnasko et al., 2010). Despite evidence of co-neurotransmitter release, it is uncertain 

how DA/glutamate co-release may contribute to GID behavior. 

A promising phenomenon that could provide a logical functional explanation for 

the role of DA/glutamate release in GID is known as vesicular synergy. The general idea 

behind vesicular synergy involves a loading enhancement of non-glutamate 

neurotransmitters into secretory vesicles via the co-localization of a vesicular glutamate 

(VGLUT) protein (El Mestikawy et al., 2011). This phenomenon has been well 

documented in cholinergic neurons: the presence of vesicular glutamate transporter 3 

(VGLUT3) and vesicular acetylcholine transporter (VAChT) on the same synaptic 

vesicle enhances packaging and release of acetylcholine (Gras et al., 2008). Vesicular 

synergy in other systems, however, such as DA and GABAergic neurons, has only 

recently begun to be explored (see (Prévost et al., 2024, 2025)). In dopaminergic 

systems, it is hypothesized that, if VGLUT2 protein and VMAT2 are present on the same 

synaptic vesicle, enhanced packaging of DA will occur and lead to increased DA release 

(Aguilar et al., 2017; Hnasko et al., 2010; H. Shen et al., 2018). The data described 

above in VGLUT2 knock-out mice supports the occurrence of this phenomenon in DA 

neurons (Hnasko et al., 2010; H. Shen et al., 2021).  

If VMAT2/VGLUT2 are co-localized on the same vesicle in grafted DA neurons, 

increased DA release could potentially lead to the development of GID. While 

promising, current clinical trials have exhibited varying support behind increased DA 

69 

 
release and GID behavior (see above); however, researchers are limited by the proper 

tools for analysis. Although postmortem evidence of asymmetric synapses formed by 

grafted DA neurons in PD subjects (T. Freund et al., 1985; Kordower et al., 1997; 

Mahalik et al., 1985; Mercado et al., 2021; Soderstrom et al., 2008) support this idea, 

future experiments are still needed to definitively determine whether VMAT2 and 

VGLUT2 are found on the same vesicle in the grafted striatal environment. As will be 

discussed in later portions of my thesis, my preclinical evidence suggests that the 

theory of vesicular synergy provides a compelling mechanism of how DA/glutamate co-

transmission (and excessive DA release) could underlie GID behavior. Furthermore, as 

mentioned above, GIDs are a complex behavioral malady in which not only one 

mechanism is likely responsible. The theory of vesicular synergy provides one 

parsimonious explanation for both the phenomenon of excess DA and the phenomenon 

of abnormal graft-host synaptic circuitry.  

Overall, the manifestation of aberrant GID behavior in a subpopulation of PD 

patients who received VM transplants has limited neural grafting as an effective 

therapeutic approach for PD. While promising preclinical and clinical studies have 

investigated the possible underlying mechanisms of GID behavior, its true pathogenesis 

remains elusive, as does the solution to its successful amelioration. Most recently, and 

for many reasons, clinical grafting trials have begun utilizing different cell sources 

including induced pluripotent stem cells (iPSCs). Because VM transplants are the only 

cell source to demonstrate GID induction thus far, clinical outcomes of iPSC grafting 

trials remain a gap in our knowledge, adding another obstacle toward optimization of 

cell transplantation therapy for patients with PD.  

70 

 
Alternative Cell Sources  

To date, the most successful cell source in neural transplantation is eVM tissue 

that contains developing SN DA neurons. Collectively, the studies addressed above 

have provided sufficient evidence that transplanted eVM tissue can survive long term, 

successfully produce DA, and induce behavioral improvement in individuals with PD. 

However, using this cell source is not without caveat: utilizing eVM tissue is 

encumbered with several practical and ethical concerns. Ethically, the use of eVM 

neurons from aborted tissue is highly controversial and not accepted in many countries 

(Brundin et al., 2010). As a practical concern, trying to procure sufficient amounts of 

tissue (approximately 4-10 embryos per patient) on a nation-wide scale is nearly 

impossible (Barker et al., 2017; Stoddard-Bennett & Reijo Pera, 2019) and contributes 

further to the immunological concerns of multiple allograft donors. Indeed, in the 

TRANSEURO clinical grafting trial, only 20 of the planned 90 surgeries were conducted 

due to low tissue supply (human embryonic ventral mesencephalic, hEVMs) (Barker et 

al., 2017).  

To combat these issues, a number of different cell sources are being investigated 

as alternatives to eVM tissue. Some of these alternative sources include neural stem 

cells and bone marrow mesenchymal cells. Most recently, the field has shifted its 

attention to the use of human pluripotent stems cells (hPSCs), which include human 

embryonic stem cells (hESCs) and iPSCs, as promising cell sources in clinical grafting 

trials. Therefore, a brief discussion on each source can be found below.  

71 

 
Figure 1.10: hESCs vs. iPSCs as cell transplantation sources for PD.  
Two  sources  of  dopaminergic  progenitors  currently  being  utilized  as  cell  sources  for 
transplantation in PD include human embryonic stem cells (hESCs) and human induced 
pluripotent  stem  cells  (iPSCs).  hESCs  are  harvested  from  human  blastocysts  and 
differentiated  into  midbrain  dopaminergic  progenitor  cells  (mDAPs)  for  transplantation. 
iPSCs  are  reprogrammed  from  somatic  cells  (e.g.,  fibroblasts)  from  adult  donors, 
differentiated into mDAPs, and then transplanted into the patient’s brain. Adapted from 
(Parmar  et  al.,  2020). Abbreviations:  hESCs  =  human  embryonic  stem  cells;  mDAPs: 
midbrain dopaminergic progenitor cells (mDAPs); iPSCs = induced pluripotent stem cells. 

Human Embryonic Stem Cells (hESCs) 

Human embryonic stem cells (hESCs) are derived from pre-implantation 

embryos and can be successfully differentiated into authentic midbrain dopaminergic 

neurons (Figure 1.10) (Barker et al., 2017). In 1998, Thompson and colleagues 

reported the first successful hESC derivation, stimulating interest in the use of hESCs 

due to its unlimited capacity for self-renewal and pluripotent differentiation (Brundin et 

al., 2010; Thomson et al., 1998). Later on, hESCs were shown to successfully survive 

and provide functional benefit following engraftment into mouse, rat, and non-human 

72 

 
 
primate models of PD (Kirkeby et al., 2012; Kriks et al., 2011; Roy et al., 2006). Most 

importantly, hESCs were found to be molecularly and functionally identical to human 

eVM DA neurons (Grealish et al., 2014; Parmar et al., 2020). In a 2017 trial of 

parkinsonian non-human primates that received intrastriatal hESC-transplants, 

behavioral improvement was demonstrated for at least 24 months following 

engraftment, and there were slight increases in DA which correlated with behavioral 

improvement (Cyranoski, 2017). These findings provided preclinical data for a phase 

I/IIa ESC-based clinical transplantation study in China, although results from the trial are 

not yet available (Wang et al., 2018).  

The current limitations of utilizing hESCs as a cell source for transplantations in 

PD include the necessity for immunosuppression and the possibility for tumorigenesis 

(Stoddard-Bennett & Reijo Pera, 2019). Because of the unlimited capacity for self-

renewal, hESCs have the ability to differentiate into various somatic cell types. In this 

way, it is possible for hESCs to form teratomas in the host brain; thus, particular care 

must be taken in order to avoid differentiation into non-neuronal cells (Brundin et al., 

2010). Also with hESCs, strong immunosuppressants must be administered prior to 

transplant surgery to avoid graft rejection and human leukocyte antigen (HLA) matches 

are required.  

Human Induced Pluripotent Stem Cells (iPSCs) 

Induced pluripotent stem cells (iPSCs) are another source being investigated as 

an alternative to eVM neuron transplants. iPSCs can be generated by reprogramming 

differentiated cells taken from the patient (e.g., fibroblasts) into an embryonic state 

(Figure 1.10) (J. Takahashi, 2018; K. Takahashi et al., 2007; K. Takahashi & Yamanaka, 

73 

 
2006) and then are pushed directly into DA neurons (Doi et al., 2014; Hargus et al., 

2010; Rhee et al., 2011; Swistowski et al., 2010; Theka et al., 2013). Using iPSCs, 

which come from non-embryonic tissue, removes the ethical obstacles that are present 

with eVM neurons. Additionally, the need for postoperative immunosuppressants is 

greatly reduced because iPSCs permit HLA matches (i.e., autologous transplantation).  

Several preclinical animal studies of PD have demonstrated that transplanted 

human iPSC-derived DA neurons can survive long-term and enable functional motor 

benefit (Doi et al., 2014; Hargus et al., 2010; Kikuchi et al., 2011; Rhee et al., 2011; 

Swistowski et al., 2010). Human iPSCs were also successfully transplanted into 

parkinsonian non-human primates, revealing robust growth, proliferation, and 

integration two years following surgery (Kikuchi et al., 2017). These studies led to the 

first clinical trial of iPSC-derived DA neurons grafted into patients with PD held in Kyoto, 

Japan in 2018. Since this trial, only one additional human case-study has been 

conducted. Autologous iPSC-derived DA neuron transplants were engrafted into a 

single individual with PD; the cells survived two years, but there were no significant 

changes in the patient’s MDS-UPDRS Part III scores. However, the patient did show an 

improvement in the Parkinson’s Disease questionnaire 39 (PDQ-39) (Schweitzer et al., 

2020).  

Unfortunately, one of the greatest limitations of using iPSCs for neural 

transplantation is the cost: reprogramming a patient’s cells may be not only a lengthy 

process but also expensive for the patient (Stoddard-Bennett & Reijo Pera, 2019). 

Moreover, like hESCs, iPSCs also possess a substantial proliferative capacity. 

Therefore, incomplete, or uncontrolled differentiation is possible, increasing the potential 

74 

 
risk for tumor formation in the grafted neurons (Brundin et al., 2010; J.-Y. Li, 

Christophersen, et al., 2008). To date, neither in the non-human primate study nor the 

Kyoto clinical trial have iPSC transplants resulted in tumor formation; however, the use 

of iPSCs is only in its infancy, so further research is warranted. Please see Table 1.1 for 

a list of the current planned or ongoing clinical trials that are utilizing hESCs or human 

iPSCs for transplantation in patients with PD.  

Additional Limitations of Cell Transplantation Therapy 

Although still a promising alternative therapeutic for PD, like most therapies, cell 

transplantation is not without limitation. While the overall goal of neural transplantation 

in PD is to repair the loss of dopaminergic neurons by engrafting new ones, this will not 

“cure” PD. In many cases, drug treatment and rehabilitation will still be required 

following transplantation surgery (Mishima et al., 2021). Additionally, this method cannot 

holistically treat all signs and symptoms of PD: non-motor dysfunction remains following 

transplantation as these symptoms stem from various other pathways in the brain 

(Barker et al., 2024).  

Not only are non-motor symptoms not targeted with neural transplantation, motor 

recovery in response to DA grafts can also be variable. Variability has been 

demonstrated both between different clinical trials and among individuals within the 

same trial (Barker et al., 2013; Winkler et al., 2005). Following engraftment, some 

patients have demonstrated great graft-derived motor benefit, while others have 

exhibited limited to no benefit (e.g., (Freed et al., 2001)). For example, one individual 

with PD who received an embryonic VM DA neuron transplant demonstrated dramatic 

recovery for 12 years after engraftment. However, by year 18, graft-derived motor 

75 

 
benefit was almost non-existent for this patient, despite robust graft survival and 

extensive innervation (W. Li et al., 2016). Postmortem analysis of another patient who 

received an embryonic VM DA graft revealed a significantly dense and widespread 

graft; however, the patient never experienced motor benefit and had to receive DBS for 

GID (Kordower, Goetz, et al., 2017).  

Another shortcoming of this experimental therapy concerns the development of 

α-synuclein pathology within the grafted cells. Α-synuclein-positive protein inclusions 

were found to develop in human embryonic VM midbrain tissue engrafted into patients 

with PD 10+ years post-transplantation (Barker et al., 2024; Kordower et al., 2008; J.-Y. 

Li, Englund, et al., 2008). It was reported that α-synuclein in these grafts was 

phosphorylated at Serine residue 129, indicative of disease-related, aggregated α-

synuclein (Anderson et al., 2006; J.-Y. Li, Englund, et al., 2008). Statistically, in the Li et 

al. report, only 1.9% of a patient’s 12-year-old graft contained Lewy bodies—a  number 

that increased to approximately 5% in another patient with a 16-year-old graft (J. Li et 

al., 2010). Some studies have found no Lewy pathology in long-term grafts up to 14 

years old (Hallett et al., 2014; Mendez et al., 2008). While potentially problematic, these 

data argue that only a small portion of the transplanted dopaminergic neurons will 

develop PD pathology, and despite the presence of pathological α-synuclein inclusions, 

some patients have still demonstrated motor benefit. Therefore, researchers believe 

that the presence of this pathology should not invalidate cell transplantation as a 

therapy for PD.  

Scientists remain uncertain why and how Lewy pathology occurs in grafted 

neurons, theorizing that pathology spreads to the transplanted neurons via a prion-like 

76 

 
mechanism (Brundin et al., 2010; Brundin & Kordower, 2012; Brundin & Melki, 2017; 

Kordower & Brundin, 2009; J.-Y. Li, Englund, et al., 2008; Olanow & Brundin, 2013; 

Olanow & Prusiner, 2009; Surmeier et al., 2017) or that α-synuclein is upregulated and 

aggregated in response to inflammation (Brundin et al., 2010). Indeed, α-synuclein 

pathology has been known to transfer from host to graft in parkinsonian mouse and rat 

models (Hansen et al., 2011; Kordower et al., 2011). Although the significance of α-

synuclein pathology in neural transplantation has yet to be determined, it will be critical 

to consider with the future use of autologous stem cell transplants as there may be a 

possibility of pathology spread from the host to donor cells (Parmar et al., 2020).  

Lastly, a limitation that has emerged more recently is the lack of a standard 

surgical device used to stereotaxically deliver DA cells to the brain. For instance, it is 

thought that the variety of different devices used in the original human embryonic VM 

transplant trials could have exacerbated the negative outcomes or heterogeneity in 

clinical responsiveness. Without a regulated global standard surgical device, developing 

a consistent, effective transplantation protocol will be challenging. Therefore, deciding 

on a device for cell implantation will have to be carefully considered moving forward, 

especially with stem cell trials commencing, in order to greatly reduce heterogeneity in 

clinical outcomes (Barker et al., 2024).  

As the field of regenerative medicine continues to evolve, especially with the 

increasing use of stem cells in ongoing clinical trials, it will be imperative to continue to 

carefully consider the limitations and concerns surrounding cell therapy. As addressed 

above, the aberrant side effect of GID remains a significant obstacle, and understanding 

its underlying pathology will be necessary for this field to continue to advance. While it is 

77 

 
true that a large number of factors have been addressed in prior clinical grafting trials 

(e.g., disease severity, patient age, and removal of 5-HT neurons), other factors that 

could affect patient outcomes in response to transplantation remain relatively 

unexplored (e.g., genetic risk factors). Ultimately, PD is a disease of complex 

heterogeneity. Therefore, moving toward a precision-medicine-based approach could be 

crucial in effectively developing and optimizing therapies that will provide maximum 

benefit for each and every patient, particularly in the context of neural transplantation.  

78 

 
 
 
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CHAPTER 2: ADVANCING CELL-BASED THERAPY FOR PARKINSON’S DISEASE 
THROUGH THE SCOPE OF PRECISION MEDICINE 

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UNDERSTANDING THE COMPLEXITY OF PATIENT RESPONSE TO PD THERAPY 

Introduction to Precision Medicine 

Precision medicine, also referred to as personalized medicine, is a conceptual 

framework that aims to tailor treatment for an individual based on his or her 

characteristics (Collins & Varmus, 2015; Schneider & Alcalay, 2020). While the 

traditional approach is to prescribe one established treatment for all patients (Figure 

2.1a), using a precision medicine approach considers an individual’s biology, 

environment, and lifestyle when developing or prescribing treatment (Figure 2.1b). 

Precision medicine may additionally focus more specifically on genetic profiles, cell 

types, biomarkers, and molecular pathways in order to achieve the most effective 

therapeutic intervention for the patient (Collins & Varmus, 2015; Payami, 2017). One 

long-term goal of precision medicine, especially for neurodegenerative disease, is to 

diagnose a patient at the earliest stages of the disease so that the proper, most effective 

treatment can be initiated as soon as possible. As clinical medicine continues to 

advance, using a precision medicine approach will likely evolve from a 

diagnosis/treatment focus to more of an emphasis on prevention of disease.  

While precision medicine is not a new concept, it has recently begun to be put 

into practice more regularly in healthcare. Just a decade ago, in 2015, the Precision 

Medicine Initiative (PMI) was launched in the United States by former President Barack 

Obama. The NIH awarded this initiative approximately $55 million in order to build its 

infrastructure so that advances could be made toward a new era of precision medicine 

(Payami, 2017). The advancements we have made since then with technologies such 

as genome sequencing, pharmacogenetics, Big Data, and artificial intelligence (AI) have 

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drastically accelerated our progress of implementing a precision-medicine-based 

approach to clinical care and preclinical research. A powerful example of the impact that 

precision medicine has had thus far is demonstrated in the field of oncology: oncologists 

work to identify anatomical spread, biology, and possible genetic changes that could 

have triggered the growth of cancer cells in a specific patient (Espay et al., 2017; Sherer 

et al., 2016). In this way, scientists and doctors have been able to develop precise, 

successful treatments based on certain characteristics of a patient’s cancer. 

Precision Medicine in Parkinson’s Disease 

Although precision medicine in oncology has had substantial success, precision 

medicine approaches for other diseases and disorders such as PD require more 

attention. Unlike oncology, one of the challenges in neurology is the limited availability 

of tissue biopsies for histological and biochemical analyses of individual patients. This, 

unfortunately, makes it difficult to identify biomarkers for neurological and 

neurodegenerative diseases (Keller et al., 2012; Schalkamp et al., 2022). Specifically, in 

PD, personalized medicine has not yet been fully realized largely due to the immense 

heterogeneity in the clinical manifestation of the disorder (Mishima et al., 2021). Among 

the 9.3 million people who live with PD worldwide, age of onset, rate of progression, and 

severity of symptoms vary dramatically, even in individuals who have the same 

mutations in at-risk genes (e.g., LRRK2) (Espay et al., 2017; Maserejian et al., 2020; 

Schalkamp et al., 2022). The ultimate problem for PD, then, is trying to get one 

treatment to work for all patients (Payami, 2017). 

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Figure 2.1: Precision medicine in Parkinson’s disease (PD). 
(a) The traditional approach to treating all patients with PD. This is considered a “one-
size-fits-all approach in which, despite differences in age of onset, disease severity, sex, 
patients  receive  similar  pharmacological  interventions  (e.g.,  levodopa  or  dopamine 
agonists).  With  this  approach,  only  a  small  population  of  patients  will  demonstrate 
significant efficacy of the prescribed therapeutic. Others may develop adverse reactions, 
and another subpopulation of patients may experience no benefit or detriment at all. (b) 
Examples of a precision-medicine-based approach for patients with PD. Each individual 
patient may exhibit differences in genetic profiles, biomarkers, and/or molecular pathways 
and should be treated accordingly. As the scientific community continues to investigate 
the  intricacies  of  PD,  more  precise  treatments  are  being  developed  which  will  provide 
safe and effective treatments for all patients, not just a small population.  

128 

 
 
 
 
 
 
 
Heterogeneity in Clinical Response to PD-related Therapy 

A prominent example of the heterogeneous nature of PD is an individual’s 

differential response to levodopa, the mainstay pharmacological therapeutic for PD. 

Levodopa is generally effective in treating motor symptoms of PD; however, the clinical 

response for each patient remains highly variable. As mentioned previously in Chapter 

1, in early-stage PD patients who received the same dose of levodopa, responses 

ranged from a 100% improvement to a 242% worsening of UPDRS Part III scores 

(Hauser et al., 2009). This variability suggests that various biochemical mechanisms are 

involved, requiring differential treatment approaches (i.e., precision medicine) between 

patients (Stoddard-Bennett & Pera, 2019).  

Currently, the heterogeneity in PD is being extensively studied. Yet, underlying 

characteristics and mechanisms remain unclear. In line with differential responses to 

levodopa, some studies have pointed to certain mutations and/or single nucleotide 

polymorphisms (SNPs) that have been associated with side effect development from 

chronic levodopa use including LID. Specifically, carriers of a polymorphism in the DA 

active transporter 1 gene (DAT1), a gene involved in DA reuptake, are 2.5 times more 

likely to develop LID (Cacabelos, 2017; Moreau et al., 2015; Stoddard-Bennett & Pera, 

2019). Another study recounted that there was a dose-dependent association between 

a variant in the GRIN2A gene (which encodes for the NR2A subunit of the N-methyl-D-

aspartate (NMDA) glutamatergic receptor) and susceptibility to LID behavior (Ivanova et 

al., 2012).  

One of the most prominent SNPs that has been linked to differential patient 

responses to levodopa (e.g., LID) is a common SNP known as rs6265 found within the 

129 

 
gene for brain-derived neurotrophic factor (BDNF) (see (Martinez-Carrasco et al., 2023) 

for other SNPs). In a retrospective analysis conducted by Fischer and colleagues, 

rs6265-carriers (aka heterozygous Val/Met or homozygous Met/Met) who received 

levodopa monotherapy reported worse UPDRS scores compared to wild-type (Val/Val; 

WT) subjects (~6 points worse) (Fischer et al., 2020). Met-allele carriers also exhibited a 

higher risk of developing LID earlier in treatment in contrast to their WT counterparts 

(Fischer et al., 2020; Foltynie et al., 2009). Alternatively to levodopa monotherapy, 

however, unmedicated Met-allele carriers presented a lower severity of motor symptoms 

compared to WT patients. Disease progression was slower for unmedicated Met-allele 

carriers, confirmed by a delayed need for levodopa (Fischer et al., 2018). Met-allele 

carriers also had a 5.3-year later age of onset of PD (Białecka et al., 2014; 

Karamohamed et al., 2005a).  

ROLE OF BDNF IN HETEROGENETIY OF CLINICAL RESPONSE TO PD THERAPY 

Large portions of this section were reproduced from (Szarowicz et al., 2022) with 

permission from the publisher.  

Current research has added to our understanding of the global risk factors (e.g., 

age, disease severity) of cell transplantation (see Chapter 1). However, the role of 

specific genetic variations remained entirely unexplored until recent studies conducted 

by our group which focused on the rs6265 SNP in the BDNF gene (see (Mercado et al., 

2021, 2024). Because of promising evidence of the role of rs6265 in heterogenetic 

responses to levodopa therapy, and the biological relevance of BDNF (detailed below), 

our laboratory utilized a precision-medicine-based approach to investigate whether 

rs6265 was a risk factor that impacts therapeutic efficacy of DA neuron transplantation 

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therapy for PD. Structure, function, and significance of BDNF as a critical neurotrophic 

factor is first discussed in-depth below.  

Introduction to BDNF 

BDNF is a neurotrophin that functions to regulate and promote neuronal survival, 

differentiation, and outgrowth of central and peripheral neurons (Gonzalez et al., 2016; 

Kowiański et al., 2018; Liu et al., 2018; Park & Poo, 2013; Sasi et al., 2017; Urbina-

Varela et al., 2020; Zagrebelsky et al., 2020). Other members of the mammalian 

neurotrophin family include nerve growth factor (NGF), neurotrophin 3 (NT-3), and 

neurotrophin 4/5 (NT-4/5), and they share more than a 50% sequence homology in their 

primary structure with BDNF (Al-Qudah & Al-Dwairi, 2016). NGF was the first 

neurotrophin to be discovered by Rita Levi-Montalcini and Viktor Hamburger in the 

1950s (Levi‐Montalcini & Hamburger, 1951, 1953). Using chick embryos, their work 

described the observation that neurons die when they lack contact with their targets; 

research which led to their later revelation that the target was a critical source of a 

diffusible growth factor eventually identified as NGF (Levi‐Montalcini & Hamburger, 

1951, 1953). In 1982, a few decades following this discovery, BDNF was isolated by 

Yves-Alain Barde and Hans Thoenen from pig brain (Barde et al., 1982). Their research 

demonstrated that this novel growth factor could induce neuronal outgrowth and survival 

of cultured embryonic chick sensory neurons (Barde et al., 1982), supporting the 

“neurotrophic hypothesis” developed by Levi-Montalcini and Hamburger (Levi‐Montalcini 

& Hamburger, 1951). Although BDNF had a similar molecular weight to NGF, its 

functional capacities were distinct, and NGF neutralizing antibodies were not able to 

block its survival-promoting activity (Levi‐Montalcini & Hamburger, 1953). Follow-up 

131 

 
cloning experiments established the identity of BDNF with a unique sequence and 

structure (Leibrock et al., 1989). 

Nearly all brain regions have been reported to contain BDNF at varying 

concentrations, but its specific function depends on stage of development as well as the 

composition of neuronal, glial, and vascular constituents present in the anatomical 

region (Kowiański et al., 2018). BDNF is abundant in the cortex, hippocampus, and 

visual cortex. It is also found in the STR, the SN, and ventral tegmental areas (VTA), 

though BDNF found in the STR is supplied by cortical and nigral DA neuron afferent 

projections and not the local neurons themselves (Baydyuk & Xu, 2014). This trophic 

factor is not solely abundant in the central nervous system (CNS) but is also released in 

appreciable amounts in the peripheral nervous system (PNS) and by other non-

neuronal cells including lymphocytes, microglia, megakaryocytes, endothelial cells, and 

smooth muscle cells (Brigadski & Lessmann, 2020). BDNF production and signaling is 

critical for a vast array of neurophysiological processes including, but not limited to, 

neuronal survival, dendritic spine development, synaptogenesis, neurite outgrowth, 

neuroprotection, long-term potentiation (LTP), and long-term depression (LTD) (for 

review (Gonzalez et al., 2016; Kowiański et al., 2018; Park & Poo, 2013; Sasi et al., 

2017; Zagrebelsky et al., 2020)). BDNF has also been found to be a necessary factor in 

neurogenesis and osteogenesis in human bone both in vitro and in vivo (Liu et al., 2018; 

Urbina-Varela et al., 2020). 

BDNF Gene Structure and Isoform Processing 

The human BDNF gene is located on chromosome 11p13-14 and is composed of 

multiple noncoding exons and one coding exon. There are 11 exons that can be 

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alternatively spliced to produce a minimum of 17 transcripts, but each transcript 

generates the same final protein product (Aid et al., 2007; Cattaneo et al., 2016; Vaghi 

et al., 2014). Of the 11 exons, 9 fall within the 5’ region (Notaras & van den Buuse, 

2019). The BDNF messenger ribonucleic acid (mRNA) transcripts that contain exons II 

and VII are exclusively expressed in the brain, whereas the transcripts containing exons 

I, IV, and V are expressed in peripheral tissue; exons VI and IX are broadly expressed 

(Urbina-Varela et al., 2020). BDNF transcription terminates at two polyadenylation sites 

within exon IX, thus giving rise to two distinct mRNA populations including short (0.35 

kb) or long (2.85 kb) 3’ untranslated regions (UTR) (Cohen-Cory et al., 2010; Notaras & 

van den Buuse, 2019; Urbina-Varela et al., 2020). These two distinct populations have 

differing localizations: short UTR BDNF (exon I and IV) transcripts are found in the cell 

soma, whereas long UTR BDNF transcripts (exon II and IV) are trafficked to dendrites to 

regulate dendritic morphology and affect LTP (Chiaruttini et al., 2009; Notaras & van 

den Buuse, 2019). 

The major coding sequence of BDNF is present in exon IX at the 3’ end and is 

translated into an inactive precursor polypeptide (i.e., preproBDNF) in the rough 

endoplasmic reticulum (ER) (Brigadski & Lessmann, 2020; Cattaneo et al., 2016; 

Pruunsild et al., 2007). Within the rough ER, the signal sequence is immediately 

cleaved to yield the 28- to 32-kDa isoform proBDNF (Brigadski & Lessmann, 2020; 

Notaras & van den Buuse, 2019) which is comprised of an N-terminal prodomain and C-

terminal mature domain (Figure 2.2a). Post-translational modifications including N-

linked glycosylation of the prodomain, as well as sulfation of the N-linked 

oligosaccharides, can take place as the proBDNF neurotrophins migrate from the Golgi 

133 

 
apparatus to the trans-Golgi network (TGN). The processing of proBDNF continues via 

cleavage by intracellular proteolytic enzymes in the TGN (i.e., furin) or by convertases 

present in intracellular secretory vesicles for extracellular export (Pang et al., 2016). A 

portion of full-length proBDNF proteins is also released and can subsequently bind the 

high affinity receptor, p75NTR (R. Lee et al., 2001). After release from the cell, 

extracellular processing of proBDNF by plasmin or matrix metalloproteases (e.g., MMP-

2, MMP-9) can also occur (Figure 2.2b) (Brigadski & Lessmann, 2020; R. Lee et al., 

2001; Mizoguchi et al., 2011; Pang et al., 2016). Processing of the preproBDNF yields 

three distinct active isoforms: the ~30kDa proBDNF, the ~13kDa mature BDNF 

(mBDNF), and the ~17kDa BDNF pro-peptide (McGregor & English, 2019) (Figure 2.2).  

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Figure 2.2: BDNF Gene Structure, Processing, and Secretion. 
a) Schematic representation of human BDNF gene structure and isoforms. Grey boxes 
represent exons; exon IX (blue) contains the major coding sequence of BDNF (Brigadski 
& Lessmann, 2020; Cattaneo et al., 2016; Pruunsild et al., 2007). b) Following translation 
into  preproBDNF  in  the  ER,  the  signaling  sequence  is  cleaved,  and  proBDNF  is 
transported through the Golgi apparatus to the trans-Golgi network. Here, proBDNF can 
be cleaved by intracellular proteolytic enzymes sorting into the constitutive or regulated 
pathways  (Brigadski  &  Lessmann,  2020;  Pang  et  al.,  2016).  ProBDNF  can  also  be 
cleaved  within  the  vesicles  or  extracellularly,  generating  mBDNF  and  the  BDNF  pro-
peptide  (McGregor  &  English,  2019).  c)  The  common  SNP  rs6265  (aka  Val66Met)  is 
located within the prodomain region of the BDNF gene and results a substitution of valine  

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Figure 2.2 (cont’d)  
(Val) for methionine (Met) at codon (G/A) 66. (Baj & Tongiorgi, 2009; Colucci-D’amato et 
al.,  2020).  Abbreviations:  pro-peptide  =  cleaved  BDNF  pro-peptide;  mBDNF/BDNF  = 
mature BDNF; proBDNF = BDNF isoform with pro-domain and mature domain. 

BDNF Sorting and Release 

Two distinct pathways of secretion exist for proBDNF and mBDNF: the 

constitutive and the regulated pathways. The constitutive pathway involves packaging 

BDNF into small-diameter granules that release BDNF independently of calcium 

fluctuation (Al-Qudah & Al-Dwairi, 2016). The majority of BDNF is packaged for release 

via the regulated pathway into larger granules that fuse to the plasma membrane in 

response to a calcium-dependent trigger (Figure 2.2b). Thus, the regulated release of 

BDNF occurs during activity-dependent depolarization (Al-Qudah & Al-Dwairi, 2016; 

Brigadski & Lessmann, 2020; Lessmann & Brigadski, 2009; Wong et al., 2015) (Figure 

2.2b). Proper sorting and secretion of BDNF is critical for the maintenance of synaptic 

plasticity, neuronal survival, and CNS homeostasis (Al-Qudah & Al-Dwairi, 2016; 

Brigadski & Lessmann, 2020; Cunha et al., 2010; Mizui et al., 2015). As such, disruption 

of BDNF sorting and/or secretion has been implicated in various neurodegenerative and 

psychiatric diseases. While the specific molecular mechanisms associated with 

improper BDNF secretion remain largely uncertain, current evidence correlates 

reductions of hippocampal and cortical volumes (Frodl et al., 2006), formation of 

abnormal synapses (Mercado et al., 2021), and decreases in dendritic complexity (Z. Y. 

Chen et al., 2006; Egan et al., 2003) as consequences of dysfunctional BDNF sorting 

and reduced secretion. 

For the regulated pathway, two binding interactions drive sorting of BDNF into 

vesicles. The BDNF prodomain/pro-peptide region binds directly to either sortilin, a 

136 

 
vacuolar protein sorting 10 (Vps10) domain-containing molecule, or carboxypeptidase E 

(Brigadski & Lessmann, 2020; Notaras & van den Buuse, 2019). Sortilin contains a 

transmembrane region and a cytoplasmic tail responsible for signaling endosome 

sorting in the Golgi apparatus (Notaras & van den Buuse, 2019). Sortilin and BDNF 

have been observed to colocalize within large dense-core vesicles, and sortilin 

truncation mutations result in impaired sorting of BDNF to the regulated pathway, 

subsequently decreasing activity-dependent release (Z. Y. Chen et al., 2005). Similarly, 

membrane-bound carboxypeptidase E is a glycoprotein that binds BDNF, and 

knockdown of carboxypeptidase E in mice has also demonstrated a reduction of 

downstream activity-dependent BDNF release (Lou et al., 2005; Notaras & van den 

Buuse, 2019). After being sorted into large dense-core vesicles of the regulated 

pathway, BDNF is generally trafficked to the axon where it can be degraded by the 

lysosome (Evans et al., 2011) or secreted into the synaptic cleft in response to neuronal 

activation where it can activate two classes of receptors, TrkB and p75NTR (defined 

below) (Carvalho et al., 2008; Lu et al., 2014; Skaper, 2018). While the majority of 

BDNF is transported anterogradely, approximately 23% of BDNF is retrogradely 

transported to dendrites, although the biological significance of its retrograde trafficking 

has yet to be elucidated (Adachi et al., 2005; Dieni et al., 2012; Notaras & van den 

Buuse, 2019). 

BDNF Signaling 

Neurotrophins are known to bind to two classes of receptors: a tropomyosin 

receptor kinase (Trk) and a pan neurotrophin receptor (p75NTR) which is a member of 

the tumor necrosis factor super family (Reichardt, 2006) (Figure 2.3). More specifically, 

137 

 
proBDNF binds with high affinity to p75NTR (R. B. Meeker & Williams, 2015; Reichardt, 

2006) (Figure 2.3a). In contrast, mBDNF preferentially binds to its high affinity receptor, 

TrkB, following its release into the synapse (Carvalho et al., 2008; Skaper, 2018) 

(Figure 2.3b). While mBDNF can also bind p75NTR, it does so with low affinity (Binder & 

Scharfman, 2004). Additionally, the BDNF prodomain/pro-peptide region binds directly 

to sortilin, thereby participating in proper sorting of this molecule to its regulated 

pathway (Z. Y. Chen et al., 2005). 

proBDNF and p75NTR 

ProBDNF binds to p75NTR upon release, stimulating nuclear factor kappa B (NF-

κB), c-Jun N-terminal Kinases (JNKs), and Ras homolog family member A (RhoA) 

signaling that modulate survival, apoptosis, and growth cone motility, respectively (M. V. 

Chao, 2003; Kowiański et al., 2018; Reichardt, 2006; Teng et al., 2005) (Figure 2.3a). 

The specific cascade that is activated is dependent on which receptors are complexed 

with p75NTR. For instance, when complexed with sortilin, pro-apoptotic pathways are 

activated (Friedman, 2000; R. B. Meeker & Williams, 2015). Recent evidence indicates 

that signaling through p75NTR can also synergistically aid in TrkB activation (Hempstead, 

2006; R. Meeker & Williams, 2014; Zanin et al., 2019). Specifically, p75NTR can 

heterodimerize with TrkB, increasing TrkB binding affinity for mBDNF, thus promoting 

neuronal growth and survival (R. B. Meeker & Williams, 2015; R. Meeker & Williams, 

2014; Zanin et al., 2019). 

mBDNF and TrkB 

Upon mBDNF binding to full-length TrkB, TrkB dimerizes and autophosphorylates 

several of its tyrosine kinase residues including Y705 and Y706 in the cytoplasmic loop 

138 

 
of the kinase domain, as well as Y515 and Y816 (Diniz et al., 2018; Notaras & van den 

Buuse, 2019). Multiple signaling pathways can be triggered once TrkB is activated 

including the phosphatidylinositol 3-kinase (PI3K), the phospholipase-C-γ1 (PLC-γ1), 

the guanosine triphosphate hydrolases of RhoA, and the mitogen-activated protein 

kinase (MAPK)/Ras cascades (reviewed in (M. V. Chao, 2003; Reichardt, 2006; Segal, 

2003)). The PI3K pathway engages in pro-survival activity and enhances dendritic 

growth and branching (Jaworski et al., 2005; Kumar et al., 2005). The MAPK/Ras 

signaling cascade controls protein synthesis during neuronal differentiation (Molina & 

Adjei, 2006). Lastly, growth of neuronal fibers is activated via activation of RhoA (Figure 

2.3b) (Kowiański et al., 2018; Reichardt, 2006). 

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Figure 2.3: Schematic representations of conventional proBDNF and mBDNF 
signaling cascades. 
a) ProBDNF binds with high affinity to p75NTR, initiating downstream JNK, RhoA, and NF-
kB  signaling  (M.  V.  Chao,  2003;  Kowiański  et  al.,  2018;  Reichardt,  2006;  Teng  et  al., 
2005).  b)  mBDNF  binds  with  high  affinity  to  TrkB,  inducing  its  dimerization  and 
autophosphorylation  and  activating  three  main  signaling  pathways,  PI3K,  PLCγ,  and 
Ras/MAPK,  all  of  which  lead  to  activation  of  the  transcription  factor  CREB,  driving 
transcription  of  genes  crucial  for  neuronal  growth  and  survival  (Mitre  et  al.,  2017; 
Reichardt,  2006;  Segal,  2003).  RhoA  signaling  and  mTOR  pathways  can  also  be 
activated leading to growth cone modulation and translation of proteins involved in the 
regulation of cellular proliferation (Diniz et al., 2018; Kumar et al., 2005; R. B. Meeker & 
Williams, 2015; Notaras & van den Buuse, 2019). 

140 

 
 
 
 
 
It is widely accepted that the proBDNF and mBDNF ligands induce opposing 

outcomes through their preferential binding to different receptors in order to promote 

neurological homeostasis (Kowiański et al., 2018). Specifically, mBDNF-TrkB signaling 

stimulates neuronal growth and synaptic plasticity, whereas signaling through 

p75NTR tends to initiate apoptosis thought to be important in development for eliminating 

inessential neurons (Friedman, 2000; Teng et al., 2005). Moreover, while mBDNF 

signaling is instrumental in driving hippocampal LTP, proBDNF promotes LTD 

(Deinhardt et al., 2011; Sakuragi et al., 2013; Woo et al., 2005; Yang et al., 2014). 

Because of homeostatic regulation, the expression of p75NTR and TrkB are known to be 

tightly linked where they are co-expressed on the surface of the cell to establish 

signaling between cell survival and cell death (Notaras & van den Buuse, 2019). 

Homeostasis can therefore be disrupted when there is an imbalance in the expression 

of these receptors or an imbalance in the levels of proBDNF and mBDNF isoforms. For 

example, research conducted by Suelves and colleagues (Suelves et al., 2019) 

examined the consequences of BDNF/TrkB/p75NTR imbalance in a Huntington’s disease 

(HD) mouse model, showing that the reduction of BDNF and TrkB levels, along with an 

increase in p75NTR expression, correlated with striatal neuropathology and motor 

dysfunction. Pharmacological normalization of p75NTR levels rescued neuropathology 

(e.g., dendritic spine density) and motor deficits (Brito et al., 2013; Suelves et al., 2019). 

In addition to changes in receptor levels/balance, increased proBDNF levels 

have been correlated with adverse outcomes in neurodegenerative disorders. 

Specifically, in mice expressing one BDNF allele with a mutated cleavage site, 

hippocampal proBDNF levels rose and promoted a decrease in dendritic arborization as 

141 

 
well as hippocampal volume (Diniz et al., 2018; Yang et al., 2014). Further reinforcing 

the importance of homeostatic balance in brain health, in PD, serum levels of proBDNF 

have been reported to be significantly higher in individuals with early PD as compared 

to heathy controls, whereas mBDNF levels were significantly lower (X. Yi et al., 2021). 

Collectively, an abundance of data indicate that tight control of both BDNF ligands and 

their receptors is critical for proper neuronal function and/or survival. 

BDNF Pro-Peptide and Sortilin 

It has been demonstrated that BDNF pro-peptide binding to sortilin drives proper 

sorting of BDNF into vesicles of the regulated secretory pathway (Z. Y. Chen et al., 

2005). In addition, the BDNF prodomain (pro-peptide), once cleaved from proBDNF, 

appears to function as an independent ligand similar to proBDNF and mBDNF isoforms 

(Anastasia et al., 2013; Mizui et al., 2016, 2017). Upon cleavage from proBDNF and its 

subsequent release, the BDNF pro-peptide binds to sortilin and complexes with p75NTR, 

resulting in various effects on the BDNF signaling cascade, neuronal survival, and 

synaptic plasticity (Anastasia et al., 2013; Z. Y. Chen et al., 2005; Giza et al., 2018; 

Mizui et al., 2016, 2017), although specific mechanisms and downstream pathways 

remain to be elucidated. 

PD and BDNF 

While dysfunction in BDNF signaling is not considered a primary cause of PD, it 

has long been known to be important for survival and development of SNpc DA neurons 

(Hyman et al., 1991; Yurek & Fletcher-Turner, 2001). In addition, there is abundant 

literature demonstrating that, in the aged brain, there is diminished BDNF, diminished 

upregulation in response to stress, reduced expression of several BDNF transcription 

142 

 
factors, and decreased expression of its TrkB receptor (for review (Mercado et al., 

2017)). Given that the primary risk factor for PD is aging, and given the critical role of 

BDNF in the well-being of SNpc DA neurons, BDNF dysfunction has been abundantly 

explored in PD. 

Current evidence has demonstrated reduced expression of BDNF mRNA 

transcripts in the SNpc in PD (Howells et al., 2000; Murer et al., 2001) as well as lower 

levels of BDNF protein specifically in the SN of individuals with PD compared to other 

brain regions, and significantly reduced serum BDNF (Scalzo et al., 2010). In addition to 

decreases in BDNF transcript levels, Scalzo and colleagues (Scalzo et al., 2010) have 

demonstrated that decreased BDNF levels are also detectable in serum of individuals 

with PD compared to healthy individuals and that concentrations were correlated with 

PD symptom severity (Scalzo et al., 2010) (Figure 2.4a). However, as the disease 

progresses, BDNF levels have been shown to increase (Knott et al., 2002; Scalzo et al., 

2010; Ventriglia et al., 2013), thought to be a compensatory mechanism in later disease 

states. 

In addition to changes in BDNF in PD, expression of TrkB receptors, which have 

high expression in SNpc neurons (Jin, 2020), has been shown to be altered in 

individuals with PD with evidence of isoform-specific alterations. For instance, levels of 

truncated TrkB have been reported to decrease in axons of the striatum, whereas levels 

were reported to increase in the striatal soma and distal dendrites of the SN in 

individuals with PD (Fenner et al., 2014). Full-length TrkB levels, in contrast, were found 

to be decreased in striatal neurites and in the cell soma of dendrites, but levels were  

143 

 
Figure 2.4: Summary of altered BDNF expression levels and consequences of the 
rs6265 SNP in neurodegenerative and psychiatric disorders. 
(a) Decreased BDNF mRNA and protein expression in various regions of the brain in PD 
(Baquet et al., 2004; Howells et al., 2000; Y. Huang et al., 2018; Razgado-Hernandez et 
al., 2015; Scalzo et al., 2010), AD (Hock et al., 2000; Narisawa-Saito et al., 1996; Peng 
et  al.,  2005;  Phillips  et  al.,  1991),  HD  (Ferrer  et  al.,  2000;  Knott  et  al.,  2002),  MDD 
(Dwivedi et al., 2003; Januar et al., 2015; Lima Giacobbo et al., 2019; Molendijk et al., 
2014; Pandey et al., 2008; Shimizu et al., 2003), and schizophrenia  (Hashimoto et al., 
2005; Reinhart et al., 2015; Weickert et al., 2003, 2005; Xiu et al., 2009). (b) Associations 
of rs6265 SNP expression and disease state including therapeutic efficacy, age of onset, 
and  susceptibility  to  the  disease:  PD  (Drozdzik  et  al.,  2014;  Fischer  et  al.,  2018; 
Karamohamed et al., 2005b; Sortwell et al., 2021), AD (Borroni et al., 2009; Fukumoto et 
al.,  2010;  Laing  et  al.,  2012),  HD  (Alberch  et  al.,  2005),  MDD  (Hosang  et  al.,  2014; 
Losenkov et al., 2020; Pei et al., 2012), Schizophrenia (H. M. Chao et al., 2008; Gratacòs 
et al., 2007; Kheirollahi et al., 2016; Suchanek et al., 2013; Z. Yi et al., 2011). (c) BDNF 
replacement strategies currently being implemented preclinically and clinically (reviewed 
in (Zuccato & Cattaneo, 2009)).  

144 

 
 
higher  in  cell  somas  and  axons  of  the  striatum  and  SNpc,  respectively  (Fenner  et  al., 

2014; Mitre et al., 2017). These findings are corroborated in mouse models of PD where 

reduced levels of BDNF protein in the SNpc results in a reduction in DA neurons as well 

as a subsequent decrease in striatal DA (Baquet et al., 2004; Porritt et al., 2005). Further, 

haplo-insufficiency of the BDNF receptor, TrkB, in transgenic mice has been associated 

with degeneration of SNpc DA neurons over time and in association with aging (for review 

(Mercado et al., 2017)). 

Utilizing BDNF as a Potential Therapeutic  

Overall, BDNF levels are negatively correlated in neurodegenerative and 

psychiatric disorders (Figure 2.4a). Therefore, many BDNF-targeted therapies aim to 

raise the levels of BDNF either exogenously or endogenously. Exogenous application of 

BDNF through direct infusion has been demonstrated to be beneficial to varying 

degrees in numerous animal studies (Altar et al., 1994; Arancibia et al., 2008; Deng et 

al., 2016; Hung & Lee, 1996). As a neuroprotective agent in PD models against DA 

neuron toxins such as 6-OHDA or MPTP, BDNF is effective at protecting SH-SY5Y 

neuroblastoma neurons in vitro and can modestly protect against 6-OHDA in vivo (Altar 

et al., 1994). Despite promising outcomes from select research conducted in preclinical 

animal models, a large-scale clinical trial involving oral BDNF supplementation at 

dosages of 50–100 mg/day in patients with amyotrophic lateral sclerosis (ALS) did not 

significantly increase patient survival (Bradley, 1999). In a clinical trial involving 

intrathecal delivery of BDNF to ALS patients, doses of 150 mg/day were well tolerated; 

however, conclusions about treatment efficacy were unable to be drawn due to small 

sample sizes (Ochs et al., 2000). However, a later trial also using intrathecal BDNF for 

145 

 
ALS found a lack of clinical efficacy (Kalra et al., 2003). These disappointing clinical trial 

results could, in part, be due to the poor pharmacokinetics of BDNF.  

The pharmacokinetics of neurotrophins are complex, making BDNF 

administration for brain therapeutics especially difficult. Neurotrophins are large, sticky 

molecules that cannot readily cross the blood-brain-barrier, have short half-lives 

reported to be 30 min or less (Habtemariam, 2018), inefficiently diffuse into tissues 

(Zuccato & Cattaneo, 2009), and approaches like intrathecal delivery result in broad 

exposure to nontargeted structures, thus limiting their scope of effectiveness (Zuccato & 

Cattaneo, 2009). If pharmacokinetic barriers could be overcome, consideration needs to 

be given to therapeutic concentrations of BDNF intended for delivery as well as the 

availability and status of TrkB receptors. Specifically, exogenous administration of BDNF 

in regions with significant reductions in TrkB expression, which is known to occur in PD 

and AD, could severely limit therapeutic benefit. In addition, excessive levels of BDNF 

could also have a negative impact. Not only can higher concentrations of BDNF 

downregulate TrkB expression, but excessive amounts of BDNF can lead to unwanted 

side effects such as seizures, fever, weight loss, fatigue, and diarrhea (Mitre et al., 

2017). Molecularly, excess BDNF can likewise have a negative effect on synaptic 

circuitry, learning, and memory by inducing hyper-excitation in regions such as the 

hippocampus (Yeom et al., 2016). Keeping the above challenges in mind, non-

pharmacological methods of BDNF delivery bear potential. 

BDNF Gene- and Cell-Based Therapy 

A promising non-pharmacological therapeutic technique is in vivo BDNF gene de-

livery. This technique involves utilizing viral vectors to transduce host cells with the 

146 

 
BDNF gene for downstream endogenous in situ mRNA and protein production. In this 

way, the high concentrations of local BDNF production in specific regions will ideally 

protect degenerating neurons in diseases such as PD, HD, and AD (Nagahara & 

Tuszynski, 2011). Preclinically, in a post-stroke depression rat model, intranasal delivery 

of a BDNF-encoding adeno-associated viral vector (AAV-BDNF) increased BDNF mRNA 

and protein in the prefrontal cortex, alleviating depressive-like symptoms (C. Chen et 

al., 2020). Additionally, preventative intrastriatal injections of AAV-BDNF reduced the 

loss of NeuN, a pan neuronal maker, in a lesioned rat model of HD, therefore providing 

neural protection (Kells et al., 2004). Although clinical trials of gene therapy that 

intended to supplement another neurotrophic factor (i.e., GDNF or neurturin) for 

neuroprotection against PD have been conducted, results are not yet promising 

(Manfredsson et al., 2020; Marks et al., 2010; Merola et al., 2020). Moreover, it remains 

unknown if it is clinically viable to target low BDNF levels in neurodegenerative or 

psychiatric disorders via gene therapy. 

Another available BDNF-targeting gene therapy involves an ex vivo autologous 

approach for neuroregeneration. Briefly, cells such as fibroblasts are taken from the 

subject, genetically modified to produce BDNF, and then transplanted back into the cell 

donor’s brain. Like in vivo methods, this strategy could allow for the sustained release of 

BDNF locally in specific brain regions but advantageously would be poised to avoid 

immune rejection. Levivier and colleagues showed that genetically modified fibroblasts 

were able to prevent degeneration induced by 6-OHDA in a rat model of PD (Levivier et 

al., 1995). Likewise, in a quinolinic acid toxin model of HD, rat fibroblasts were 

genetically engineered to produce BDNF and transplanted back into the rat brain, 

147 

 
resulting in the protection of striatal neurons as compared to control animals (Kells et 

al., 2004). Similarly, mesenchymal stem cells (MSCs) genetically altered to overexpress 

BDNF have been shown to reduce neuropathological and behavioral deficits in rodent 

models of HD, suggesting that these approaches have considerable potential for clinical 

use (for review (Crane et al., 2014)). 

Gene therapy, whether viral vector-mediated or autologous transplantation of 

genetically modified cells, holds strong promise but is not without caveats (Baum et al., 

2003, 2004). In general, local release of BDNF is difficult to tightly regulate genetically, 

and as introduced above, overproduction of BDNF can be detrimental to the circuitry of 

the brain (Yeom et al., 2016; Zuccato & Cattaneo, 2009). In addition, both approaches 

involve invasive surgical protocols; however, in the scope of neurosurgery methods that 

are much more aggressive (e.g., tumor resection), the approach for vector or cell graft 

delivery is minimally invasive and straightforward. Of additional concern is immune 

response to viral vectors and the associated products of foreign transgenes (Bulaklak & 

Gersbach, 2020). However, as recently reviewed, current efforts and advances in 

clinical trials have led to advances to circumvent immune obstacles including modifying 

AAV capsids to evade pre-existing neutralizing antibodies and development of new 

methods for clearing of antibodies from circulation (for review (Bulaklak & Gersbach, 

2020)). With the advent of new DNA modification techniques, it is not beyond the realm 

of possibilities that novel gene therapy approaches could be applied in the future. In 

addition, given that ex vivo autologous treatment was well tolerated, and symptom 

improvement was demonstrated in AD (Nagahara & Tuszynski, 2011; Tuszynski et al., 

148 

 
2005), this approach remains hopeful to those suffering from neurodegenerative or 

neurological disorders. 

BDNF Mimetics 

One of the most promising BDNF-related administration strategies involves the 

use of BDNF mimetics. These are small molecules designed to mimic the binding loops 

of BDNF, resulting in the phosphorylation and activation of TrkB and its downstream 

effectors, AKT and ERK (Du & Hill, 2015; Kazim & Iqbal, 2016; Zuccato & Cattaneo, 

2009). The use of small molecules allows for the delivery of controlled dosages with 

improved pharmacokinetics compared to full-length BDNF. Mimetics have shown 

improved diffusivity, blood-brain-barrier permeability, and augmented receptor specificity 

with less promiscuity (Cardenas-Aguayo et al., 2013; Du & Hill, 2015; Kazim & Iqbal, 

2016; Zainullina et al., 2021). These compounds, however, would require repeat dosing 

and would not be brain region-specific in targeting, potentially trafficking to areas where 

their engagement is not advantageous (Kazim & Iqbal, 2016; Longo & Massa, 2013). 

Two common BDNF mimetics are 7,8-dihyrodxyflavone (DHF) and GSB-106. 

7,8-DHF is a naturally occurring flavonoid responsible for binding and initiating TrkB 

signaling pathways. 7,8-DHF application has been investigated in many 

neurodegenerative and neurological disorders including PD and AD (Bollen et al., 2013; 

Devi & Ohno, 2012; Jang et al., 2010). For example, in a comprehensive report by Jang 

and colleagues (Jang et al., 2010), 7,8-DHF was documented in mice to specifically 

activate TrkB in the brain, to diminish kainic acid-induced toxicity in the hippocampus, to 

decrease infarct volumes in a middle cerebral artery occlusion model of stroke, and it 

was neuroprotective in a MPTP model of Parkinson’s disease (Jang et al., 2010). 

149 

 
Additionally, in a mouse model of AD, cognitive deficits were restored after 7,8-DHF 

administration (Bollen et al., 2013; Devi & Ohno, 2012). Collectively, these studies 

support the idea that 7,8-DHF may be a therapeutic mimetic worth implementing in a 

wide range of disorders. 

Another common mimetic is bis-(N-monosuccinyl-L-seryl-L-lysine) hexameth-

ylenediamide, also referred to as GSB-106, and it mimics the interaction between the 

TrkB receptor and BDNF via loop 4 of BDNF. Like 7,8-DHF, GSB-106 administration 

elicits neuroprotective properties by preventing apoptosis in SH-SY5Y cells through the 

suppression of caspase-3 activity (Zainullina et al., 2021). As reviewed in (Gudasheva, 

Povarnina, et al., 2021), GSB-106 has also been shown to have a variety of TrkB-

mediated neuroprotective effects as well as reduce depressive-like symptoms in a 

mouse model of depression where administration increased locomotor activity and 

reduced signs of anhedonia (Gudasheva, Povarnina, et al., 2021; Gudasheva, 

Tallerova, et al., 2021). Studies focused on these two BDNF mimetics demonstrate that 

these small molecules represent potentially useful treatment approaches for those with 

neurodegenerative diseases such as PD. Continued preclinical and clinical 

development are needed so that their therapeutic effects can be optimized to the 

greatest extent. 

Diet and Exercise 

Diet and exercise are widely accessible, non-invasive, low-cost treatments that 

are of interest for neurodegenerative and neurological conditions. Preclinical studies in 

various animal models confirm that dietary and exercise regimens increase BDNF levels 

in the brain and improve cognitive and behavioral functions (Duan et al., 2001; 

150 

 
Fahnestock et al., 2012; Maswood et al., 2004; Mattson et al., 2002; Zuccato & 

Cattaneo, 2007, 2009). For example, Fahnestock and colleagues (Fahnestock et al., 

2012) demonstrated that implementing a diet high in antioxidants in aged dogs 

increased BDNF(Fisher et al., 2008; Herman et al., 2007; Stuckenschneider et al., 

2016) transcripts to levels which were comparative to the young dog cohort (Fahnestock 

et al., 2012). Additionally, restricting the diet of 3-month-old male Sprague Dawley rats 

to an alternate day feeding regimen compared to ad libitum increased BDNF levels in 

multiple brain regions including the cortex, striatum, and hippocampus (Duan et al., 

2001). There also is a wealth of data suggesting that exercise provides neuroprotection 

in multiple animal models of PD (Fredriksson et al., 2011; Lau et al., 2011; Petzinger et 

al., 2007; Tajiri et al., 2010; Toy et al., 2014; Tuon et al., 2012; Wu et al., 2011) with 

additional indications that it improves motor symptoms and quality of life in individuals 

with PD. Studies using heterozygous deletion of BDNF (Gerecke et al., 2012) or 

inhibition of BDNF TrkB receptors (Real et al., 2013) demonstrate that BDNF is 

essential for the beneficial effects of exercise on the neuroprotection of the nigrostriatal 

DA system in PD rodent models. 

In patients with depression, exercise was found to induce significant increases in 

serum levels of BDNF levels in all assessed participants (Szuhany et al., 2015). After 

sprint interval training, BDNF levels were increased directly afterward, then returned to 

baseline within 90 minutes in eight male subjects (Reycraft et al., 2020). A number of 

genes, including BDNF, are associated with risk for post-traumatic stress disorder 

(PTSD) (Voisey et al., 2019). Intriguingly, in combat veterans with PTSD, active exercise 

reduced methylation of the BDNF gene at specific CpG sites, resulting in normalized 

151 

 
gene expression of BDNF as compared to those without active exercise (Voisey et al., 

2019). Although there are many studies reporting that diet and exercise lead to 

increased BDNF levels (Duan et al., 2001; Fahnestock et al., 2012; Reycraft et al., 

2020), the specific mechanisms responsible have yet to be elucidated. In the context of 

BDNF as a therapeutic target, understanding and harnessing the benefits of diet and 

exercise on BDNF function could lead to vital non-invasive treatments geared toward 

improving not only neurodegenerative or psychiatric conditions but general patient 

quality of life. 

Genetic Polymorphisms of BDNF 

Remarkably, more than one hundred polymorphisms have been described in the 

BDNF gene (Tudor et al., 2018; Urbina-Varela et al., 2020). While many known variants 

exist within non-coding regions, understanding of their functional consequences 

remains limited. However, the most extensively studied SNP is the Val66Met (G196A, 

rs6265) polymorphism within the prodomain region of the BDNF gene. Other less well-

studied variants exist within this region including Thr2I1e (rs8192466), Gln75His 

(rs1048221), Arg125Met (rs1048220), and Arg127Leu (rs1048221) and are reviewed 

elsewhere (R. Huang et al., 2007; Notaras & van den Buuse, 2019; Shen et al., 2018; 

Urbina-Varela et al., 2020). 

rs6265 (Val66Met) 

The rs6265 BDNF SNP, or Val66Met, results from a nucleotide exchange from 

guanine to adenine at position 196 (G196A). This change results in a substitution of 

valine to methionine at codon 66, thus referred to as Val66Met (Anastasia et al., 2013) 

(see Figure 2.2c). An individual can be heterozygous (Val66Met) or homozygous 

152 

 
(Met66Met) for this SNP. The prevalence of this SNP worldwide is approximately 20%, 

with certain populations in East Asia reporting an incidence up to 72% (Mercado et al., 

2021; Petryshen et al., 2010; Tsai, 2018). Found in the prodomain region of the BDNF 

gene, this substitution creates binding interference between the BDNF prodomain/pro-

peptide of proBDNF to sortilin. The consequential result, and subsequent hallmark of 

this polymorphism, is a decrease in activity-dependent release of BDNF, with no 

reported alterations in constitutive release (Egan et al., 2003; Urbina-Varela et al., 

2020). The reduction in BDNF release is dose-dependent with homozygous subjects 

showing significantly less release compared to heterozygous subjects (Met/Met > 

Val/Met > Val/Val) (Mercado et al., 2021). 

Several studies have documented a variety of neuropathologies associated with 

the decrease in secreted mBDNF linked to rs6265 including reduction of hippocampal 

and cortical volume, abnormal synaptic connections, and decreased dendritic 

complexity and arborization (Z. Y. Chen et al., 2006; Chiaruttini et al., 2009; Egan et al., 

2003; Frodl et al., 2006; Y. Lee et al., 2013; Mercado et al., 2021). The functional 

consequences of this common genetic variant are wide-reaching and have been 

documented to impact memory and cognition, anxiety, and depression, and have been 

associated with obsessive compulsive disorder (OCD), attention deficit hyperactivity 

disorder (ADHD), schizophrenia, multiple sclerosis (MS), blepharospasm, and migraines 

(Cai et al., 2017; Z. Y. Chen et al., 2006; Di Carlo et al., 2019; Egan et al., 2003; Frodl et 

al., 2006; Y. Lee et al., 2013; Mei et al., 2022; Shang et al., 2022; Siokas et al., 2019). 

Such pathology may be linked to evidence demonstrating that the BDNF Val66Met 

substitution can result in binding disruption of the translin/trax complex to BDNF mRNA 

153 

 
transcripts, subsequently compromising transport of transcripts to dendrites which is 

critical for synaptic plasticity and dendritic complexity (Chiaruttini et al., 2009; Cohen-

Cory et al., 2010; Notaras & van den Buuse, 2019). As a consequence, decreased 

BDNF trafficking to dendrites may have negative implications in multiple 

neurodevelopmental and neurological disorders (Chiaruttini et al., 2009; Di Carlo et al., 

2019). 

In addition, decreased BDNF levels/signaling (i.e., rs6265) have been implicated 

in several neurodegenerative disorders including AD, PD, and HD (Figure 2.4b). How 

the expression of this common human genetic variant impacts PD is highlighted below 

as the aforementioned brain maladies (e.g., AD, HD) are beyond the scope of this thesis 

research. Please see Figure 2.4 for more details regarding BDNF in these other 

neurodegenerative and neurological disorders.  

rs6265 and PD 

Although expression of the BDNF rs6265 Met allele is not correlated with an 

increased incidence of PD, it may contribute to worsening non-motor symptomology 

(Fedosova et al., 2021; Gorzkowska et al., 2021; Shen et al., 2018). For example, 

apathy is one of the most common non-motor neuropsychiatric symptoms of PD 

(Gorzkowska et al., 2021), and although not statistically significant, PD individuals who 

were homozygous for the Met allele (i.e., Met/Met) were reportedly more likely to display 

apathetic emotions compared to those without the Met/Met genotype. Moreover, the 

risks of impulsive-compulsive and related behavioral disorders are also statistically 

correlated in individuals with PD when expressing the rs6265 SNP (Fedosova et al., 

2021). 

154 

 
An important distinction of Met allele carriers with PD has been in their response 

to certain pharmacotherapies including levodopa treatment (see full discussion above in 

the “Heterogeneity” section). Specifically, it has recently been reported that Met allele 

carriers, homozygous or heterozygous, reported worse UPDRS scores when 

administered levodopa monotherapy compared to their homozygous Val allele carrier 

counterparts (Fischer et al., 2020; Sortwell et al., 2021). Individuals expressing the Met 

allele were also found to have a higher risk of developing the often debilitating side 

effect known as LID earlier in their treatment compared to homozygous Val allele 

carriers (Drozdzik et al., 2014; Foltynie et al., 2009). 

To contrast these negative correlations of the Met allele, in unmedicated PD 

patients, a lower severity of motor symptoms has been observed in the initial stages of 

the disease in BDNF variant individuals (Fischer et al., 2018). Although homozygous 

Met-allele carriers tended to have more tremor-like symptoms, the progression of the 

disease was slower, with delayed need for levodopa administration compared to Val 

allele carriers (Fischer et al., 2018). Along with this notable decrease in severity of 

motor symptoms, a later age of onset of PD was reported in homozygous Met allele 

individuals compared to their Val/Val and Val/Met counterparts with one cohort reporting 

a 5.3-year later age of onset (Białecka et al., 2014; Karamohamed et al., 2005a) 

(Figure 2.4b). In contrast, Svetel et al., 2013 reported that the presence of the Met 

allele was not associated with clinical characteristics of PD including age of onset and 

disease severity (Svetel et al., 2013). 

155 

 
HETEROGENEITY IN SIDE EFFECT LIABLITY OF CELL TRANSPLANTATION 

GID and the rs6265 BDNF SNP  

As discussed previously in Chapter 1, a subpopulation of patients developed 

graft-induced dyskinesia (GID) as a side effect following primary DA neuron 

transplantation in clinical trials for PD (Freed et al., 2001; Olanow et al., 2003). Now, 

after decades of rigorous preclinical research following the enacted moratorium in the 

early 2000s (Hagell & Cenci, 2005), several clinical grafting trials for PD are now 

planned or ongoing (Barker et al., 2019); example clinical trial identifiers NCT04802733, 

NCT01898390, NCT03309514, NCT03119636, NCT04146519). A comprehensive list of 

the current planned/ongoing clinical cell transplantation trials are listed in Table 1.1 in 

Chapter 1. While these experiments have strived to optimize patient selection (i.e., age, 

disease severity, cell preparation) prior to transplantation (Barker et al., 2024), the 

underlying mechanisms of aberrant GID behavior remain, to this day, unknown. Until 

GIDs are addressed, neural grafting for PD will not be considered a safe or optimized 

therapeutic option for PD patients. For a comprehensive discussion of the postulated 

mechanisms underlying GID behavior, please see Chapter 1.  

Goals of Current Research 

Because the underlying mechanisms of GID remain a gap in our knowledge, taking 

the necessary actions to fully understand its underlying pathology is the first step in 

developing a precision-medicine approach for neural therapy. The rs6265 SNP, which 

has been implicated in clinical outcomes for levodopa treatment (Fischer et al., 2020), 

and now cell-based therapy (e.g., (Mercado et al., 2021, 2024), points to the rationale 

for continuing research in this area. Therefore, the overarching hypothesis for my 

156 

 
dissertation research centers around a probable role for the rs6265 SNP in the 

underlying mechanisms responsible for the substantial heterogeneity demonstrated in 

grafted patients with PD (i.e., GID development). Indeed, while my predecessor, Dr. 

Natosha Mercado, successfully demonstrated that DA-grafted homozygous rs6265 

(Met/Met) parkinsonian rats exhibit enhanced functional recovery following engraftment 

of WT DA neurons (i.e., earlier and more robust amelioration of LID), she conversely 

demonstrated that DA-grafted Met/Met parkinsonian rats uniquely develop aberrant GID 

compared to WT subjects (Mercado et al., 2021). In order to further her investigations 

into the benefit and detriment of the rs6265 SNP, I endeavored to: 

(1) examine additional host/donor genotype combinations and their impact on graft-

derived efficacy and side effect liability (i.e., GID) (Chapter 3) and 

(2) investigate whether exogenous intrastriatal administration of BDNF would 

replenish the decreased BDNF release in rs6265 Met/Met carriers, induce 

maturation/integration of grafted DA neurons, and ameliorate GID (Chapter 4). 

Considering that BDNF plays a crucial role in proper synapse formation and 

maturation of DA neurons (Gonzalez et al., 2016; Kowiański et al., 2018; Liu et al., 

2018; Park & Poo, 2013; Sasi et al., 2017; Urbina-Varela et al., 2020; Zagrebelsky et al., 

2020), it is biologically reasonable to hypothesize that aberrant and/or immature 

synaptic connectivity between host and donor, permitted by a decrease in activity-

dependent BDNF release (i.e., rs6265), underlies GID behavior. Specifically, as 

introduced in Chapter 1, Soderstrom and colleagues previously demonstrated that GID 

development in DA-grafted parkinsonian rats was associated with atypical, asymmetric 

(presumed glutamatergic) synaptic connections made by the grafted DA neurons 

157 

 
(Soderstrom et al., 2008). Moreover, Dr. Mercado further showed that the DA-grafted 

Met/Met parkinsonian rats that developed GID behavior demonstrated expression of 

vesicular glutamate transporter 2 (VGLUT2) in grafted DA neurons, indicative of an 

immature graft phenotype, and showed immunohistochemical evidence of atypical 

glutamatergic synapse formation.  

Using these findings as a basis for my thesis research, I will provide evidence in the 

next two chapters demonstrating that the homozygous rs6265 (Met/Met) genotype, 

whether found in the host or donor, confers a degree of graft-derived benefit; however, 

the Met/Met parkinsonian hosts engrafted with WT DA neurons remain the only 

host/donor combination to exhibit significant GID behavior (Chapter 3). Additionally, I will 

also demonstrate that, contrary to my hypothesis that BDNF supplementation would 

promote graft maturation and reduction of GID, BDNF supplementation instead 

exacerbated GID behavior in the Met/Met hosts engrafted with WT DA neurons 

(Chapter 4). Finally, my research provides evidence in support of the contention that 

dysregulated DA/glutamate co-transmission and/or excess DA release appear to 

contribute to GID induction.  

158 

 
 
 
 
 
 
 
 
 
 
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CHAPTER 3: PRECISION MEDICINE IN PARKINSON’S DISEASE: HOST/DONOR 
INTERACTIONS AND GRAFT-INDUCED DYSKINESIA LIABILITY IN HOMOYZGOUS 
rs6265 (MET/MET) BDNF PARKINSONIAN RATS 

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ABSTRACT 

Transplanting replacement dopamine (DA) neurons remains of worldwide interest 

as an experimental treatment for Parkinson’s disease (PD). However, like other PD 

therapies, heterogeneity in clinical responsiveness exists. To deconstruct this variability, 

our laboratory focuses on the common single nucleotide polymorphism (SNP), rs6265, 

present in the brain-derived neurotrophic factor (BDNF) gene. Our group previously 

reported that homozygous rs6265 (Met/Met) knock-in parkinsonian rats engrafted with 

embryonic wild-type (WT) DA neurons demonstrate paradoxical enhancement of graft 

function compared to their WT counterparts but uniquely develop the side effect known 

as graft-induced dyskinesia (GID). To expand our understanding of the impact of rs6265 

in DA neuron transplantation, I have examined the effect of rs6265 in both host and 

donor as part of my thesis research. Results indicate that functional benefit continues to 

occur more rapidly in the presence of the Met allele regardless of whether found in the 

host or donor. Curiously, Met/Met hosts engrafted with WT DA neurons remain the only 

group to exhibit significant GID behavior. 

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INTRODUCTION 

Parkinson’s disease is a relentlessly progressive neurodegenerative disorder that 

continues to place an immense burden on society (Dorsey et al., 2018; Straccia et al., 

2022; Yang et al., 2020). At its current growth rate, it is estimated that approximately 13 

million people will be diagnosed with PD by the year 2040 (Dorsey et al., 2018; Straccia 

et al., 2022). To treat the symptoms of PD, several pharmacological options are 

available including anticholinergic agents, DA agonists such as levodopa, monoamine 

oxidase inhibitors (MAOIs), and catechol-O-methyltransferase (COMT) inhibitors 

(Stoker & Barker, 2020). Despite the extensive competition, levodopa remains the most 

tolerated and effective pharmaceutical intervention for motor symptoms of PD, even 

after over six decades (Cotzias et al., 1967; Nutt & Wooten, 2005; Poewe et al., 2010; 

Stoker & Barker, 2020).  

Levodopa therapy, however, is not without limitations. While levodopa works well 

for PD patients for some time, individuals eventually are plagued with significant side 

effects (i.e., levodopa-induced dyskinesia (LID)) and waning efficacy as their disease 

progresses. Indeed, based on the results from a large retrospective analysis (Earlier 

versus Later Levodopa therapy in PD study; ELLDOPA), patients reported a range of 

responses to levodopa administration, from 100% improvement to a 242% worsening of 

symptoms assessed by the United Parkinson’s Disease Rating Scale (UPDRS) (Hauser 

et al., 2009). Collectively, the scientific community has recognized that PD is a 

complicated and heterogeneous disease with substantial variability in clinical 

responsiveness to existing therapeutic interventions.  

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An alternative approach aimed at mitigating the heterogenous nature of several 

PD therapies is the regenerative approach of DA neuron transplantation. Currently, the 

method that has had the most success clinically is primary embryonic ventral 

mesencephalic (eVM) DA neuron transplantation into the caudate/putamen (Olanow et 

al., 2009; Steece-Collier & Collier, 2016; Stoker et al., 2017). Unfortunately, similar to 

the heterogeneity demonstrated with levodopa administration, variability in clinical 

responsiveness also exists following cell transplantation. Further, a subpopulation of 

patients who received eVM transplants developed a novel dyskinetic side effect known 

as GID (Freed et al., 2001; Hagell et al., 2002; Olanow et al., 2003). The mechanisms 

underlying GID behavior remain unknown and controversial.  

While the field has historically gained understanding of the role of global risk 

factors (e.g., age, disease severity) in response to cellular transplantation, the role of 

genetic risk factors has been relatively unexplored until two recent studies conducted by 

our group (Mercado et al., 2021, 2024). We explored the common SNP, rs6265, found in 

the BDNF gene, which results in the decrease of activity-dependent release of BDNF 

(Chen et al., 2005; Egan et al., 2003; Urbina-Varela et al., 2020). Also referred to as 

Val66Met, the rs6265 SNP involves a valine to methionine substitution at codon 66 

(Anastasia et al., 2013). Although not correlated with an increased incidence of PD 

(Fedosova et al., 2021; Gorzkowska et al., 2021; Mariani et al., 2015; Shen et al., 

2018), rs6265 has been shown to reduce the therapeutic efficacy of levodopa in PD 

patients (Drozdzik et al., 2014; Fischer et al., 2020; Foltynie et al., 2009; Sortwell et al., 

2021). In the general worldwide population, the prevalence of rs6265 is approximately 

20%; however, in certain East Asian populations, prevalence can reach 72% (Petryshen 

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et al., 2010; Tsai, 2018). The impact of rs6265 leads to a substantial decrease in BDNF 

release (Egan et al., 2003; Urbina-Varela et al., 2020) in roughly 20% of the general 

population.  

Due to the critical role BDNF plays in promoting dendritic spine growth, synapse 

formation, and maturation of DA neurons (Gonzalez et al., 2016; Kowiański et al., 2018; 

Liu et al., 2018; Park & Poo, 2013; Sasi et al., 2017; Urbina-Varela et al., 2020; 

Zagrebelsky et al., 2020), our group previously hypothesized that the rs6265 SNP 

underlies the variability (e.g., GID) in clinical response to DA neuron transplantation in 

PD patients. Using a CRISPR rs6265 knock-in parkinsonian rat model, we 

demonstrated that homozygous rs6265 (aka Met/Met) parkinsonian rats engrafted with 

WT (Val/Val) DA neurons paradoxically exhibited enhanced neurite outgrowth and 

functional recovery compared to WT subjects. However, WT-grafted Met/Met rats 

uniquely demonstrated significant GID induction compared to WT hosts engrafted with 

cells from the same source (i.e., WT DA neurons). Interestingly, GID behavior was 

strongly correlated to expression of vesicular glutamate transporter 2 (VGLUT2), a 

marker of immature DA neurons, in Met/Met host parkinsonian rats (see (Mercado et al., 

2021)). 

Because of the relatively high prevalence of rs6265 in the general population, 

and because only WT DA neuron grafts have been studied, I endeavored to investigate 

functional outcomes of DA transplantation in both the host and donor carrying the 

rs6265 allele. Accordingly, both WT and Met/Met hosts engrafted with either WT or 

Met/Met donor neurons were studied to uncover a potentially optimal host/donor 

combination that would exhibit superior functional benefit with limited side effect liability. 

185 

 
Since enhanced functional benefit of the Met allele was reported previously (Barbey et 

al., 2014; Finan et al., 2018; Kailainathan et al., 2016; Krueger et al., 2011; McGregor et 

al., 2019; McGregor & English, 2019; Mercado et al., 2021; Qin et al., 2014; Voineskos 

et al., 2011; Zivadinov et al., 2007), we hypothesized that the Met/Met hosts and/or 

donor neurons would induce significant graft efficacy but also develop the highest GID 

severity. In this study, we report that the homozygous Met/Met genotype, whether found 

in the host or donor, produces a modest, but significant, enhanced behavioral benefit 

(i.e., earlier amelioration of LID) compared to WT hosts engrafted with WT donor 

neurons, indicating that the Met allele does indeed retain a mechanistic benefit in a DA-

grafted parkinsonian rat model similar to our first report (Mercado et al., 2021). 

Unexpectedly, the Met/Met parkinsonian recipients of WT DA grafts remained the only 

host/donor combination to develop significant GID compared to all other grafted 

host/donor groups. While a correlation between VGLUT2 expression and GID severity 

was no longer apparent as in our previous experiment (Mercado et al., 2021), evidence 

collected from this study suggests that there is a possible complex association between 

DA release and GID behavior, warranting further investigation into this phenomenon as 

a promising underlying mechanism of GID. 

186 

 
 
 
 
Animals 

METHODS 

Sprague-Dawley homozygous rs6265 (i.e., Met/Met) male rats (8-9 months at 

lesioning, 13-14 months at sacrifice) were utilized from our in-house colony derived from 

CRISPR knock-in rats generated by Cyagen Biosciences (Santa Clara, CA). These 

knock-in rats carry the valine to methionine substitution in the rat BDNF gene 

(Val68Met). Equivalent to the human Val66Met SNP, the rat rs6265 SNP is located at 

codon 68 (Val68Met) because rats have two additional threonine amino acid residues. 

Moreover, the rat BDNF gene has a 96.8% sequence homology with the human BDNF 

gene (BLAST queries: P23560 and P23363). For this study, both WT and homozygous 

rs6265 Met/Met rat hosts were employed. The Michigan State University Institutional 

Animal Care and Use Committee approved all animal experimental procedures. 

Eight animals were excluded due to failed lesion surgeries. Additional animals 

(N=5) were excluded a priori (i.e., prior to grafting) due to failure to develop effective LID 

prior to cell transplantation. Following postmortem analysis of the transplanted grafts, a 

small number (N=4) of grafted rats were excluded for having few surviving grafted cells 

(<100) or misplaced/cortically-placed grafts. One animal was excluded from analysis as 

a biological outlier (i.e., having a “hotspot” graft) (Maries et al., 2006). Final 

experimental cohorts included N=7 (WT host/sham-graft), N=8 (Met/Met host, sham-

graft), N=7 (Met/Met host, WT graft), N=9 (Met/Met host, Met/Met graft), N=7 (WT host, 

Met/Met graft), and N=6 (WT host, WT graft) (see Figure 3.1).  

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Experimental Design and Timeline 

As shown in the experimental timeline schematic (Figure 3.1), rats were 

rendered unilaterally parkinsonian by an injection of the DA neurotoxin, 6-

hydroxydopamine (6-OHDA), delivered stereotaxically to the substantia nigra pars 

compact (SNpc) and medial forebrain bundle (MFB). Two weeks following stereotaxic 

lesioning surgeries, amphetamine-mediated rotational behavioral was analyzed to 

confirm the lesion status of each animal subject. Following lesion confirmation, 

successfully lesioned rats were primed with daily (M-F) levodopa two weeks later to 

generate significant and stable LID, our primary behavioral measure of graft function 

(i.e., amelioration of LID). Levodopa priming lasted for a total of four weeks, after which 

rats underwent neural transplantation surgery. Rats received intrastriatally placed 

embryonic VM DA neurons from WT (Val/Val), rs6265 (Met/Met), or sham-grafted (cell-

free) donors. Immediately following transplantation surgeries, levodopa was withdrawn 

for one week but then reinitiated for the remainder of the experiment. For a total of 10 

weeks following engraftment, parkinsonian rats were evaluated for amelioration of LID 

behavior, rated every two weeks. At 10 weeks post-engraftment, amphetamine-

mediated rotational behavior was measured as a secondary assessment of graft 

function. As an indication of graft dysfunction, total and peak (70 minutes) 

amphetamine-mediated GID behavior was evaluated at 10 weeks 24 hours after final 

LID assessment.  

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Figure 3.1: Experimental timeline and design. 
(a)  Timeline  of  lesion  and  grafting  surgeries,  behavioral  assessment,  and  drug 
administration.  (b)  Schematic  diagram demonstrating  cell  transplantation  of  embryonic 
day 14 (E14) ventral mesencephalic (eVM) tissue from either WT (Val/Val) Sprague- 
Dawley  male  rats.  eVM  tissue  was  dissected  and  transplanted  into  either  WT  or 
homozygous  rs6265  (Met/Met)  host  parkinsonian  rats.  (c)  Experimental  schematic 
depicting  the  various  host/donor  combination  groups  following  cell  transplantation.  (d) 
Table including the genotype of the graft, donor, and final group sizes. Abbreviations: 6-
OHDA  =  6-hydroxydopaine,  amph  =  amphetamine,  LD  =  levodopa,  LID  =  levodopa-
induced dyskinesia, GID = graft-induced dyskinesia, WT = wild-type, wk = week.  

189 

 
 
 
 
 
Nigrostriatal 6-OHDA Stereotaxic Surgery 

Rats were anesthetized with 2% isoflurane (Sigma St. Louis, MO, USA) and 

positioned in a stereotaxic frame. A total of 2 μL of 6-OHDA was administered at a flow 

rate of 0.5 μL/min to the SNpc (coordinates of 4.8 mm posterior, 2.0 mm lateral, and 8.0 

mm ventral relative to bregma) and the MFB (coordinates of 4.3 mm posterior, 1.6 mm 

lateral, and 8.4 mm ventral relative to bregma). Immediately following lesion surgery, 

rats were given an intraperitoneal (i.p.) injection of 5 mg/kg carprofen (Rimadyl) for pain 

relief. Histological confirmation of successful nigral lesions was performed postmortem 

using the stereological medial terminal nucleus (MTN) DA cell enumeration method 

(Gombash et al., 2014). 

Amphetamine-mediated Rotational Behavior  

As a method to assess lesion status after stereotaxic 6-OHDA lesion surgeries, 

as well as graft function (LID) and dysfunction (GID; see below) following grafting 

surgeries, amphetamine-induced rotational behavior was employed since it is a reliable 

measure of both nigrostriatal DA depletion and function of the graft (e.g., (Collier et al., 

1999, 2015; Dunnett & Torres, 2011; Soderstrom et al., 2008)). Two weeks following 

stereotaxic lesion surgeries, amphetamine rotations were assessed to confirm lesion 

status in each rat subject. Amphetamine sulfate (2.5 mg/kg) was administered i.p. into 

each rat, and rotational behavioral was subsequently recorded for 90 minutes with the 

automated Rotameter System (TSE-Systems, Chesterfield, MO, USA). In order to be 

included for the continuation of the experiment, rats were required to rotate ≥5 ipsilateral 

rotations per minute. Additionally, at 10 weeks post-engraftment, amphetamine rotations 

were manually quantified at one-minute time intervals in the rat’s home cage at 70 

190 

 
minutes post-amphetamine injection during the assessment of GID behavior as a 

secondary readout of graft function.  

Levodopa Administration and LID Ratings  

For a total of four weeks following 6-OHDA lesion surgeries, rats were primed 

with daily (M-F) levodopa (12 mg/kg levodopa/benserazide (1:1); subcutaneous) 

administration. One week after neural transplantation surgeries, levodopa was 

withdrawn from rat subjects in order to prevent any possible toxic interactions between 

levodopa and the grafted DA neurons (Steece-Collier et al., 1990). After the one-week 

interval of no levodopa administration, levodopa was introduced again daily (M-F) 

throughout the remainder of the study.  

We employed a well-established rat LID model as a measure of graft function as 

this behavioral side effect can be improved by dopaminergic neuron grafts in both 

parkinsonian rats (Lane et al., 2006; Lee et al., 2000; Maries et al., 2006; Mercado et 

al., 2021; Soderstrom et al., 2008, 2010) and individuals with PD (Hagell & Cenci, 

2005). LIDs were assessed on days 1, 7, 14, and 21 prior to grafting, and at five post-

graft intervals including weeks 2, 4, 6, 8, and 10. The LID rating scale utilized was 

developed in our lab based on specific criteria aligned with attributes of dyskinesia (refer 

to (Caulfield et al., 2021; Maries et al., 2006)). A blinded investigator assessed LID 

behavior at one-minute intervals at 20, 70, 120, 170, and 220 minutes after levodopa 

administration, following the method previously detailed in (Mercado et al., 2021, 2024). 

A total LID severity score, determined as the sum of the severity and frequency of each 

assessed behavior, was calculated for each animal at each timepoint (Mercado et al., 

2021, 2024). 

191 

 
Donor Tissue Preparation and Neural Cell Transplantation 

After the completion of levodopa priming, rats were assigned to one of six groups 

based on pre-grafted LID severity scores. Rats were blindly and randomly assigned to a 

group in order to ensure that the average LID severity score was statistically similar 

between host/donor groups. The six host/donor combinations groups included two 

sham-grafted groups (WT or Met/Met hosts), Met/Met hosts engrafted with WT DA 

neurons (M/W), Met/Met hosts engrafted with Met/Met DA neurons (M/M), WT hosts 

engrafted with WT DA neurons (W/W) and WT hosts engrafted with Met/Met DA 

neurons (W/M). Rat hosts in each group received intrastriatal transplantations of 

200,000 VM neurons from embryonic day 14 (E14) timed-pregnant donors 

corresponding to the assigned genotype. First, the VM was harvested in cold calcium-

magnesium free (CMF) buffer, and the cells were dissociated according to our standard, 

previously reported protocol (Collier et al., 2015, and Mercado et al., 2021). Briefly, the 

tissue was incubated for 10 minutes at 37°C in CMF buffer containing 0.125% trypsin. 

Cells were then triturated with 0.005% DNase using a 2.0 mm tip Pasteur pipette, 

followed by further trituration with a sterile 3cc, 22-gauge syringe. The resulting cell 

suspension was layered onto sterile fetal bovine serum (FBS) and centrifuged at 1,000 

rpm for 10 minutes at 4°C, then resuspended in 1.0 mL of Neurobasal medium (Gibco, 

Thermo Fisher Scientific, Waltham, MA, USA). Cell number and viability were assessed 

using the trypan blue exclusion method, and the final concentration was adjusted to 

33,333 cells/μL. Cells were kept on ice throughout the surgery and transplanted within 

five hours of preparation. The cells were injected into the striatum at a single rostral-

caudal site (0.2 mm anterior, 3.0 mm lateral to bregma) and distributed at three dorsal-

192 

 
ventral (DV) locations of 5.7, 5.0, and 4.3 mm ventral to the skull base (Collier et al., 

2015; Mercado et al., 2021, 2024). A total of 2 µL of the VM cell suspension was 

injected at each DV site (injected at 0.5 µL/min), for a total of 6 µL per rat. WT and 

Met/Met sham-grafted rats received cell-free NeurobasalTM medium using the same 

injection paradigm.  

Graft-induced Dyskinesia (GID) 

Low-dose amphetamine was implemented to assess GID, with rats receiving a 

single 2mg/kg dose of amphetamine sulfate (i.p.). The amphetamine-mediated GID 

behavioral method was utilized based on evidence that DA-grafted, but not sham-

grafted, animals exhibit dyskinetic behaviors in response to low-dose amphetamine 

(Lane et al., 2009; Shin et al., 2012; Smith et al., 2012). A blinded investigator rated GID 

behavior in the same manner and using the same rating scale as described for LID 

since GID appears phenotypically similar to LID in DA-grafted rats. Amphetamine-

induced GIDs were evaluated at 10 weeks post-engraftment since GID are only notable 

upon graft maturation. GID were examined following the final LID assessment.  

The incidence of “total” GID severity and “peak” GID severity, which are both 

illustrated in Figure 3.4, are defined as the number of animals which exhibited a total 

GID severity score of 4 or higher (total) or a peak GID score of 2 or higher (peak). Peak 

GID incidence was observed at 70 minutes post-amphetamine administration. Total and 

peak GID incidence scores were determined in this way based on the fact that a score 

less than four (total) or two (peak) reflects stereotypic behavioral profiles that can occur 

in non-grafted/non-lesioned rats such as typical intermittent licking and chewing 

behavior.  

193 

 
Necropsy 

Euthanasia of the rats was achieved as detailed previously (Mercado et al., 2021 

and 2024). Briefly, rats were deeply anesthetized with phenytoin/pentobarbital (250 

mg/kg; i.p., VetOne, Boise, ID, USA) followed by intracardiac perfusions with room 

temperature 0.9% saline (heparinized) and cold 4% paraformaldehyde. Following 

intracardiac perfusion, each brain was carefully removed and placed into a 4% 

paraformaldehyde solution. The brains remained in this solution for 24 hours at 4°C. 

Next, brains were then submerged into 30% sucrose (4°C); the brains remained in 

sucrose until sectioning. When sectioned, coronal cuts of the brains were made at a 

thickness of 40 µm using a sliding microtome. Cut tissue sections were stored at -20°C 

in cryoprotectant.  

Histology 

Tyrosine hydroxylase (TH) Immunohistochemistry for Stereological Quantification 

of Graft Cell Number and Volume 

Tissue sections were rinsed in Tris-buffered saline containing 0.3% Triton-X 

(TBS-Tx), and then incubated in 0.3% hydrogen peroxide. Afterward, they were blocked 

with 10% normal goat serum (NGS) for 90 minutes. For primary antibody incubation, the 

sections were exposed overnight at room temperature to rabbit anti-TH (see Table 3.1). 

Following primary antibody incubation, the sections were rinsed, incubated with 

biotinylated goat anti-rabbit secondary antibody (Table 3.1), and then developed using 

the avidin/biotin enzyme complex and 3,3'-diaminobenzidine (DAB; 0.5 mg/mL). 

A blinded investigator employed the Stereo Investigator® Optical Fractionator 

method (MBF Bioscience, Williston, VT, USA) to quantify the number of TH-positive 

194 

 
(TH+) cells in the grafted striatum. The 20x objective (numerical aperture 0.75) was 

utilized to count cells on a Nikon Eclipse 80i microscope with a 200 µm x 200 µm 

counting frame. The optical dissector height was set to 20 µm, with a 2.0 µm guard 

zone. This process was performed on 4-12 serial (1:6) TH+ sections, with the number of 

sections varying based on the rostral-caudal spread of the graft. 

To estimate the total graft volume, a blinded investigator used the Stereo 

Investigator® Cavalieri Estimator on the same tissue sections described above. The 

central region of the graft was outlined, and a grid with random sampling points (50 µm 

spacing) was overlaid on the contours. The calculated total estimated graft volume is 

reported in mm³. 

Stereological Quantification of Neurite Outgrowth  

The Stereo Investigator® Spaceballs workflow was employed to stereologically 

determine the extent of graft-derived innervation in the host striatum. The TH+ 

immunolabeled tissue section that contained the largest portion of the graft was 

selected for analysis. Contours (345 µm x 265 µm) were manually drawn proximal and 

distal to the graft in four directions including medial, dorsal, lateral, and ventral. We 

defined the proximal region as 100 µm from the graft and the distal region measuring 

700 µm from the edge of the graft (per (Mercado et al., 2021, 2024)); this generated a 

total of eight contoured measurement sites. Spaceballs was applied to each of the eight 

contours which generated random sampling sites throughout the contour. The spherical 

probe that the Spaceballs workflow employs had a radius measuring 5.0 µm with guard 

zones of 1.0 µm. A blinded investigator collected all neurite density measurements using 

the 60x oil immersion objective on the Nikon Eclipse 80i stereotaxic microscope. The 

195 

 
numerical aperture was 1.40. Data are reported as the estimated average neurite length 

per the volume of the probe (µm/mm³) per grafted TH+ neuron (i.e., neurite density per 

grafted cell). 

Immunofluorescence (IF)  

Full series, DAB-developed TH-labeled sections as described above were used 

as a guide when choosing one representative grafted striatal section for each 

immunofluorescent and in situ hybridization assay. For all protein staining procedures, 

tissue sections were rinsed in TBS-Tx, blocked in 10% NGS/0.3% TBS-Tx, and then 

incubated overnight at 4°C. Tissue sections were then labeled with their respective 

Alexa Fluor™ secondary antibodies (1:500 dilution; see Table 3.1) for 90 minutes at 

room temperature, protected from light exposure. Sections were mounted and 

coverslipped with Vectashield® anti-fade mounting medium with DAPI (H-1500; Vector 

Laboratories, Inc. Burlingame, CA, USA). 

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Table 3.1: Targeted Antigens with corresponding antibodies. 
Secondary antibody catalog numbers are Alexa Fluor®-conjugated and purchased from 
Invitrogen®. 

Fluorescent In Situ Hybridization (FISH) using RNAscopeTM  

In order to examine the impact of the various host/donor genotypes on mRNA 

expression of the two common BDNF receptors, TrkB and p75NTR (Table 3.2) within the 

grafted DA neurons and host striatum, RNAscopeTM in situ hybridization was performed 

according to the manufacturer’s instructions for the RNAscopeTM Multiplex Fluorescent 

V2 Assay kit (Advanced Cell Diagnostics Inc., Hayward, CA, USA). Immunofluorescent 

staining for TH was followed by the completion of RNAscopeTM to stain for grafted DA 

197 

 
 
neurons. Similarly, RNAscopeTM/TH-treated tissue sections were mounted and 

coverslipped using Vectashield® anti-fade mounting medium with DAPI (H-1500; Vector 

Laboratories, Inc. Burlingame, CA, USA). 

Table 3.2: RNA Targets and RNAscopeTM probes. 

Fluorescent Image Acquisition 

All fluorescent images (1024 x 1024) of immunofluorescent and in situ 

hybridization-treated tissue sections were acquired using a Nikon A1 laser scanning 

confocal microscope system that was equipped with a Nikon Eclipse Ti microscope and 

Nikon NIS-Elements AR software. For the TrkB/p75NTR/TH protocol, the 20x 

magnification objective was employed to collect images of inside the graft, outside the 

graft, and the intact striatum of all animals. The 4x objective was used for dopamine 

transporter (DAT)/TH immunohistochemistry staining to allow collection of a full image of 

the entire graft in each striatal section. One image of the intact striatum was used for 

comparison. For both DAT/TH and TrkB/p75NTR mRNA, 2 µm z-stacks with a scan 

speed of 1/8 frame/second were taken. Likewise, 2 µm z-stacks with a scan speed of 

1/8 frame/second were taken of the tissue sections stained for Iba1/GFAP/TH. Z-stacks 

for Iba1/GFAP/TH were acquired using the 10x objective, and two images were taken to 

capture the entirety of the graft. Additional images of the intact striatum were taken for 

comparison. For the VGLUT2 colocalization inside TH+ neurons, the 60x oil-immersion 

objective (numerical aperture of 1.40) was used, and 1.5 µm-thick z-stacks were 

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acquired. The scan speed was 1/8 frame/second. Two representative images for this 

experiment were taken within the dorsolateral area of the DA graft, and one image was 

acquired of the intact striatum for comparison.  

Imaris® Fluorescent Image Quantification 

Triple-label protein mRNA analysis: TH protein and TrkB, p75NTR mRNA 

Three-dimensional (3D) images of the grafted tissue sections labeled for TrkB 

mRNA, p75NTR mRNA, and TH protein were imported into Imaris® and converted to the 

native Imaris®  file format. In order to minimize any background or off-target 

fluorescence, background subtraction was employed. A 3D surface object for the TH+ 

graft was generated using the surface function Imaris® plugin. The spots function was 

then used to select all mRNA puncta for TrkB and p75NTR both inside and outside of the 

grafted neurons. Once created, the same exact parameters were used across all 

images. The “Find Spots Close to surface” MATLAB plugin was utilized to quantify TrkB 

and p75NTR mRNA puncta within the DA graft. Data are represented as the number of 

TrkB and p75NTR mRNA puncta inside TH+ neurons (µm3), as well as total number of 

TrkB and p75NTR mRNA puncta per cell (TH+ and TH- cells) in the striatum. The ratio of 

the quantity of TrkB mRNA transcripts to the quantity of p75NTR mRNA transcripts within 

the TH+ neurons is also reported. 

Dual-label protein analysis: VGLUT2/TH 

Z-stacks of grafted tissue dual-immunolabeled for VGLUT2 and TH proteins were 

imported into Imaris® and converted into its native file format. Background subtraction 

was applied to each image to reduce background fluorescence. The surface function 

was used to generate a precise 3D reconstruction of TH+ neuron fibers within the graft 

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(µm³). The spots function was employed to detect VGLUT2 protein puncta, with 

consistent parameters applied across all images. Colocalized VGLUT2 puncta were 

then filtered through the Object-Object statistics “Shortest Distance to Surface” function 

to retain only those located within the TH surface. Data are presented as the number of 

VGLUT2 protein puncta within the grafted TH surface (µm³). 

Dual-label protein analysis: Dopamine transporter (DAT) and TH 

Confocal 2D images of dual-immunolabeled tissue for TH and DAT proteins were 

imported into Imaris® and converted into its native file format. To reduce background 

fluorescence in each channel, background subtraction was applied to all images. The 

surface function was then utilized to generate an accurate reconstruction of TH and DAT 

fibers within the graft. Data are expressed as the ratio of the sum of DAT fluorescence 

intensity to DAT surface area (µm²) relative to the sum of TH fluorescence intensity to 

TH surface area (µm²). 

Triple-label protein analysis: Iba1/GFAP/TH  

3D z-stacks of triple-immunolabeled grafted tissue sections for TH, Iba1, and 

GFAP proteins were imported into Imaris® and converted into its native file format. 

Background subtraction was applied to each image to reduce background fluorescence. 

Using semi-automatic thresholding and the surface function plugin, 3D surface objects 

were generated for TH, Iba1, and GFAP. Data are expressed as the Iba1 surface 

volume (µm³) normalized to the graft surface volume (TH; µm³), with GFAP reported 

using the same approach. 

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Statistical Analysis  

LID and GID data are created using ordinal rating scales and were statistically 

analyzed using non-parametric tests including the Kruskal-Wallis test followed by 

Dunn’s multiple comparisons, or the Mann-Whitney U tests with Dunn’s multiple 

comparisons (between subjects). Pre-graft amphetamine-mediated rotational behavior 

was analyzed using a Mann-Whitney test, and post-graft amphetamine-mediated 

rotational behavior was analyzed using the Kruskal-Wallis test with Dunn’s multiple 

comparisons. The amphetamine-mediated time course analysis at each time point (i.e., 

20, 70, 120, 170, and 220 minutes post-administration) was also analyzed using non-

parametric Kruskal-Wallis with Dunn’s multiple comparisons.  

An ordinary One-way ANOVA test with Tukey’s multiple comparisons was 

performed to analyze total enumeration and volume (µm³) of the graft. Results that were 

also analyzed using this test included quantity of VGLUT2 protein/µm³ TH, TrkB:p75NTR 

per TH neuron, DAT sum intensity/µm³, and Iba1 and GFAP volume (µm³) per TH 

neuron. An ordinary One-way ANOAV with Šidák’s post-hoc comparisons was 

conducted for average neurite density both proximal and distal to the graft. 

Mann-Whitney two-tailed tests were conducted for the total of p75NTR mRNA 

puncta inside the TH+ graft comparing the GID+ M/W host/donor group and the three 

other GID- host/donor combinations (i.e., M/M, W/W, W/M; combined based on no 

statistical differences between these GID- groups). In addition, a two-way ANOVA test 

with Tukey’s multiple comparisons was implemented to analyze the quantity of TrkB and 

the quantity of p75NTR mRNA transcripts per cell in the striatum (TH+ and TH- cells).  

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The statistical test used to compare the quantity of VGLUT2 protein per TH 

volume (µm³) between the M/W host/donor group and the combined host/donor group of 

M/M, W/W, W/M was an unpaired, two-tailed t-test to determine specific GID+ vs. GID- 

group comparisons. Also between these groups, this same statistical test was used to 

analyze the ratio of TrkB:p75NTR mRNA transcripts inside TH+ DA neurons. Unpaired, 

two-tailed t-tests were also employed for the total of TrkB mRNA puncta inside the TH+ 

graft between the GID+ M/W host/donor group and the GID- M/M, W/W, W/M combined 

groups. The DAT sum intensity/µm³ was similarly analyzed with this statistical test. 

For all correlations between protein expressions and GID behavior, a non-

parametric Spearman correlation was applied. Statistical outliers, although rate, were 

identified and removed using both the ROUT and Grubb’s outlier tests. If data met 

assumptions for normality and homogeneity of variances, parametric statistical tests 

were employed. All statistical analyses for this study were successfully completed using 

the GraphPad Prism software created for Windows (v.10.4.1).  

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RESULTS 

The homozygous rs6265 (Met/Met) genotype, in either host or donor, 

demonstrates superior graft efficacy and earlier amelioration of LID behavior 

Based on results from previous studies that revealed behavioral benefit of the 

homozygous rs6265 (Met/Met) genotype in a parkinsonian rat model (see (Mercado et 

al., 2021)), traumatic brain injury (TBI) (Barbey et al., 2014; Finan et al., 2018; Krueger 

et al., 2011), and multiple sclerosis (MS) (Zivadinov et al., 2007), I continued to 

hypothesize that the Met allele, whether found in the host or donor, would confer a 

greater degree of graft-derived benefit compared to the WT genotype in response to 

neural transplantation. I theorized that the Met/Met hosts engrafted with Met/Met DA 

neurons would exhibit the greatest degree of benefit with the earliest amelioration of LID 

behavior.  

In keeping with this hypothesis, Met/Met allele carriers, either in the host or in the 

donor neurons, exhibited enhanced behavioral recovery demonstrated by a four-week-

earlier amelioration of LID behavior. Compared to sham-grafted parkinsonian subjects, 

Met/Met hosts engrafted with WT DA neurons (M/W), Met/Met hosts engrafted with 

Met/Met DA neurons (M/M), and WT hosts engrafted with Met/Met DA neurons (W/M) 

showed significant reductions in LID behavior at week 4 post-engraftment (Figure 3.2a; 

Week 4: p = 0.0033 M/W vs. sham, p = 0.0044 M/M vs. sham, p = 0.0301 W/M vs. 

sham). In contrast, it took an additional four weeks for WT hosts engrafted with WT DA 

donor neurons (W/W) to exhibit significant amelioration of LID compared to sham-

grafted animals (Figure 3.2a; Week 8: p = 0.0266 W/W vs. sham). This significant 

difference in WT hosts engrafted with WT donor neurons, however, was lost at the 

203 

 
conclusion of week 10 for this host/donor combination (Figure 3.2a; Week 10: p = 

0.0940), while other Met-allele host/donor combinations exhibited significantly lower LID 

compared to sham-grafted animals for the duration of the study (Figure 3.2a; Week 10: 

p = 0.0003 M/W; p = 0.0202 M/M; p = 0.0217 W/M). Met-allele-carriers (host or donor) 

notably had higher percentages of improvement from pre-graft LID behavior, with the 

Met/Met hosts engrafted with WT DA neurons generating the highest percentage of 

improvement (Figure 3.2b; M/W 75.26 ± 5.67%, M/M 60.00 ± 11.37%, W/M 50.67 ± 

13.74% vs. W/W 42.60 ± 14.34%, Mean ± SEM). 

Our secondary assessment of DA neuron graft function, amphetamine-induced 

rotational behavior, demonstrated that pre-graft amphetamine-mediated ipsilateral 

rotations were not statistically different between WT or Met/Met host rats (Figure 3.2ei; 

p = 0.1465). At 10 weeks post-engraftment, similar to total LID scores, amphetamine 

rotational behavior was significantly reduced in only Met-allele carriers when compared 

to sham-grafted animals (Figure 3.2eiii; p = 0.0013 M/W; p = 0.0010 M/M; p = 0.0088 

W/M). Conversely, the number of amphetamine-mediated ipsilateral rotations in the WT 

hosts engrafted with WT donor neurons was not significantly different than sham-grafted 

subjects (Figure 3.2eiii; p = 0.0539).  

Despite enhanced recovery seen in Met-allele carriers compared to sham-grafted 

subjects, total LID scores and number of amphetamine rotations of the four host/donor 

combinations were not significantly different from each other (Figure 3.2e; p > 0.9999 

between grafted animals from Weeks 4-10 post-engraftment (LID) and p > 0.9999 at 

Week 10 (amphetamine rotations). Nevertheless, these results, along with an earlier 

reduction in LIDs (i.e., Week 4 vs. Week 8) and fewer rotations per minute at 10 weeks 

204 

 
in Met-allele carriers, support that the Met allele remains to confer a degree of benefit 

compared to the WT allele, at least in the context of neural transplantation.  

Figure 3.2: Impact of host/donor genotype on LID behavior and amphetamine-
rotational asymmetry in DA-grafted parkinsonian rats. 
(a) Total LID severity scores for each host/donor combination throughout the duration of 
the  experiment,  including  pre-  and  post-engraftment.  LID  severity  scores  were  not 
significantly different between sham-grafted groups; therefore, sham-grafted groups were 
combined post-engraftment (see inset graph separated by genotype) (p ≥ 0.0999 for all 
time points; Mann-Whitney unpaired two-tailed t-test). Statistics: Non-parametric Kruskal-
Wallis test  with  Dunn’s  multiple  comparisons  at  each  timepoint.  Week  4:  **p  =  0.0033 
M/W host/donor vs. sham-graft, **p = 0.0044 M/M vs. sham-graft, *p = 0.0301 W/M vs. 
sham-graft.  Week  6:  *p  =  0.0418  M/M  vs.  sham-graft;  Week  8:  **p  =  0.0028  M/W  vs. 
sham-graft, *p = 0.0266 W/W vs. sham-graft, *p = 0.0322 W/M vs. sham-graft; Week 10:  

205 

 a) b)  
 
 
Figure 3.2 (cont’d) 
***p = 0.0003 M/W vs. sham-graft, *p = 0.0202 M/M vs. sham-graft, *p = 0.0217 W/M vs. 
sham-graft. At no time point were the grafted groups significantly different from each other 
(p  ≥    0.3644  for  all  time  points).  (b)  Percent  improvement  in  LID  behavior  for  each 
host/donor group, from pre-graft LID scores to LID scores at 10 weeks post-engraftment. 
Statistics:  Non-parametric  Kruskal-Wallis  test  with  Dunn’s  multiple  comparisons,  p  ≥ 
0.2778  for  all  groups.  (c)  Time  course  of  LID  severity  scores  for  individual  animal 
responses at Week 4-10 following levodopa administration. Rats were rated at 20, 70-, 
120-, 170-, and 220-minutes post-injection. Statistics: Non-parametric Kruskal-Wallis test 
with Dunn’s multiple comparisons at each time point post-levodopa injection. Week 4 (20 
minutes): *p  =  0.0243 M/W vs.  sham,  **p  = 0.0097  W/M vs.  sham;  (70  minutes) **p  = 
0.0057 M/M vs. sham; (120 minutes) ***p = 0.0006 M/W vs. sham, *p = 0.0260 W/M vs. 
sham; Week 6 (20 minutes) ***p = 0.0041 M/W vs. sham, **p = 0.0066 W/W vs. sham, 
**p = 0.0028 M/M vs. sham; (70 minutes) *p = 0.0196 W/W vs. sham; Week 8 (20 minutes) 
*p = 0.0107 M/W vs. sham, **p = 0.0099 W/M vs. sham; (70 minutes) *p = 0.0195 W/W 
vs. sham; (120 minutes) **p = 0.0028 M/W vs. sham; (170 minutes) *p = 0.0235 M/W vs. 
sham, *p = 0.0132 M/M vs. sham, *p = 0.0295 W/M vs. sham; Week 10 (20 minutes) *p 
= 0.0245 M/W vs. sham, **p = 0.0091 W/M vs. sham; (70 minutes) **p = 0.0012 M/W vs. 
sham,  *p  =  0.0135  M/M  vs.  sham,  **p    =  0.0093  W/M  vs.  sham;  (120  minutes)  ***p  = 
0.0006 M/W vs. sham, *p = 0.0217 M/M vs. sham; (170 minutes) *p = 0.0228 M/W vs. 
sham.  

c) 

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Figure 3.2 (cont’d) 
(d) Total LID score for each individual animal at Week 4, 6, 8, and 10 post-engraftment. 
Statistics  (listed  within  graph):  Non-parametric  Kruskal-Wallis  test  with  Dunn’s  multiple 
comparisons at each time point. (e) Amphetamine rotational asymmetry at pre-graft and 
10 weeks post-engraftment. Data are expressed as (i) number of ipsilateral rotations per 
minute (at the 70-minute time point), and (ii, iii) average number of ipsilateral rotations 
per minute at 70 minutes  (Mean ± SEM). Statistics: (i) Mann-Whitney U unpaired two-
tailed t-test, (ii) Two-way ANOVA with Tukey’s multiple comparisons, ****p <0.0001 W/W 
vs. WT sham and W/M vs. WT sham, ***p = 0.0002 M/W vs. Met sham, ****p <0.0001 
M/M  vs.  Met  Sham.  Pre-graft  vs.  post-graft  for  all  groups  p  ≥  0.0034.  No  significant 
differences  in  rotations  were  found  between  grafted  groups,  p  ≥  0.9314.  (iii)  Non-
parametric  Kruskal-Wallis  test  with  Dunn’s  multiple  comparisons. Abbreviations:  LID  = 
levodopa-induced dyskinesia, M/M = Met/Met, LD = levodopa, ns = not significant.  

d) 

207 

 
 
 
 
Cell survival, graft volume, and neurite outgrowth are not significantly affected by 

the WT and/or homozygous rs6265 (Met/Met) genotype in host or donor  

Stereological quantification of TH immunoreactivity indicated that the estimated 

number of surviving transplanted DA neurons was not different between WT and 

homozygous rs6265 (Met/Met) host/donor combinations (Figure 3.3b; Mean ± SEM, 

M/W 2492 ± 508.9; M/M 2284 ± 294.9; W/W 2115 ± 406.7; W/M 1915 ± 219.5, p ≥ 

0.7060 for all comparisons). Likewise, graft volume (mm3) of the DA grafts was not 

statistically significant between genotypic host/donor combinations either (Figure 3.3c; 

Mean ± SEM, M/W 0.3352 ± 0.0661 mm3, M/M 0.2728 ± 0.0257 mm3, W/W 0.2231 ± 

0.0252 mm3, W/M 0.3257 ± 0.0378 mm3; p ≥ 0.2925 for all combinations).  

Previously, Met/Met parkinsonian rats paradoxically demonstrated more 

extensive graft-derived neurite outgrowth in the distal regions of the graft in contrast to 

WT host rats (Mercado et al., 2021). In a Met/Met environment, there is reduced activity-

dependent BDNF release (Egan et al., 2003), and therefore, this finding is unexpected. 

Nonetheless, based on this previous finding, I hypothesized that neurite outgrowth 

would be most extensive in the Met-allele host/donor combinations. However, all 

host/donor combinations, both proximally and distally to the graft, stereologically 

exhibited no differences in neurite outgrowth, reported as the average neurite density 

(µm/mm3) (Figure 3.3e; Proximal average: M/W vs. M/M p = 0.9667; M/W vs. W/W p > 

0.9999; W/W vs. W/M p = 0.9043; M/M vs. W/M p = 0.9986). Data also reveal no 

differences in neurite outgrowth located distal to the graft at any of the regions 

surrounding the graft (i.e., dorsal, ventral, lateral, medial) (Figure 3.3f; Distal average: 

208 

 
M/W vs. M/M p > 0.9999; M/W vs. W/W p > 0.9999; W/W vs. W/M p = 0.9967; M/M vs. 

W/M p = 0.9987).  

Figure 3.3: Impact of host/donor genotype on graft survival and neurite outgrowth 
in DA-grafted parkinsonian rats. 
(a) Histological representation of the DA-grafted parkinsonian striatum (4x) micrograph 
(Scale  bar  =  1000  µm).  (b)  Stereologically  estimated  number  of  surviving  grafted  DA 
neurons. Statistics: Mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons (c) 
Stereologically  estimated  graft  volume  (µm3).  Mean  ±  SEM.  One-way  ANOVA  with 
Tukey’s multiple comparisons. (d) Schematic depiction of grafted DA neurite outgrowth 
analysis. Proximal regions are depicted in blue, and distal regions are depicted in green. 
(e) Average neurite density of grafted DA neurons both proximal and distal to the border 
of the graft. Statistics: Mean ± SEM. Two-way repeated measures ANOVA with Šidák’s  

209 

 
 
Figure 3.3 (cont’d) 
post-hoc  test;  proximal  (p  ≥  0.8276)  and  distal  (p  ≥  0.9967).  (f)  Distal  neurite  density 
comparison between DA-grafted groups separated into each region surrounding the graft. 
Statistics: Mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons, p ≥ 0.0915 
for all host/donor grafted groups. Abbreviations: Ctx = cortex, Str = striatum, D = dorsal, 
L = lateral, M = medial, V = ventral, ns = not significant.  

e) 

f) 

Homozygous rs6265 (Met/Met) parkinsonian rats engrafted with WT DA neurons 

remain the only host/donor combination to develop aberrant GID behavior 

We previously demonstrated that homozygous rs6265 (Met/Met) parkinsonian 

rats engrafted with WT DA neurons uniquely developed aberrant GID behavior 

compared to WT rats engrafted with WT DA neurons (Mercado et al., 2021). Since the 

Met/Met genotype has a reduction in activity-dependent release of BDNF (Egan et al., 

2003), I postulated that the Met/Met parkinsonian host rats engrafted with Met/Met 

donor DA neurons (M/M) would be the host/donor combination that develops the most 

severe GID behavior compared to other host/donor combinations. In contrast, the 

parkinsonian Met/Met hosts engrafted with WT DA neurons (M/W) strikingly remain the 

only host/donor combination to develop significant GIDs (Figure 3.4). When analyzed 

against sham-grafted parkinsonian subjects, the M/W host/donor group exhibited 

210 

 
 
 
 
approximately an 8-fold increase in total GID severity (Figure 3.4a; M/W 18.21 ± 6.59 

vs. sham 2.16 ± 0.71 p = 0.0406, Mean ± SEM) and peak GID severity (Figure 3.4b; 

M/W 6.79 ± 1.61 vs. sham 0.78 ± 0.35 p = 0.0071, Mean ± SEM). Total GID severity and 

peak GID behavior was not statistically different between other DA-grafted host/donor 

combinations (Figure 3.4a,b, p ≥ 0.6774 (total); p ≥ 0.6318). 

In complement to GID severity, the incidence of GID behavior was also reported. 

Confirming the results of total and peak GID severity, percent GID incidence for total 

and peak GID was the highest in Met/Met parkinsonian rats engrafted with WT DA 

neurons (M/W) (Figure 3.4c, total GID: Mean ± SEM; sham 20.0%, M/W 71.4%, M/M 

66.7%, W/W 33.3%, W/M 42.9%; peak GID: sham 20%, M/W 85.7%, M/M 44.4%, W/W 

33.3%, W/M 28.6%). When amphetamine-mediated GID behavior at 10 weeks post-

engraftment was reported at the rating time points of 20, 70, 120, 170, and 220 minutes 

following amphetamine administration (Figure 3.4d), a statistical difference was 

prevalent at 70-minutes post-injection between the Met/Met hosts engrafted with WT DA 

grafts (M/W) and sham-grafted subjects (Figure 3.4d; M/W vs. sham, p = 0.0071, 70-

minutes). Despite no significant differences exhibited between the GID+ M/W 

host/donor combination and the other GID- DA-grafted host/donor combinations, these 

data further corroborate the findings demonstrated in total and peak GID severity 

between M/W host/donors and sham-grafted animals, ultimately signifying that only 

Met/Met parkinsonian recipients of WT donor neurons display significant aberrant GID. 

While the underlying mechanism of GID behavior remains elusive, our group 

previously demonstrated that grafted DA neurons transplanted into parkinsonian rats 

expressed morphological evidence of atypical, excitatory synapses observed with 

211 

 
positive immunoreactivity to VGLUT2, a marker of glutamatergic neurons (Mercado et 

al., 2021; Soderstrom et al., 2008). Normally, VGLUT2 is expressed in immature 

embryonic DA neurons; however, as the neurons mature, the VGLUT2 phenotype 

disappears (El Mestikawy et al., 2011). In Mercado et al., 2021, not only did the 

transplanted DA neurons retain an immature phenotype, a statistically positive 

correlation between GID severity and VGLUT2 expression was previously reported in 

the Met/Met hosts engrafted with WT DA neurons (Mercado et al., 2021). Based on this 

evidence, I endeavored to investigate, in this current study, whether all host/donor grafts 

retained an immature phenotype (i.e., VGLUT2 expression) and if VGLUT2 expression 

in the Met/Met-WT host/donor parkinsonian rats remained strongly correlated to GID 

behavior.  

Unsurprisingly, as was apparent in (Mercado et al., 2021), each host/donor 

combination (M/W, M/M, W/W, W,M) did not express statistically different quantities of 

VGLUT2 protein within the transplanted DA graft (Figure 3.4f; M/W vs. M/M p = 0.7323; 

M/W vs. W/W p = 0.9978; M/M vs. W/M p = 0.9928; W/W vs. W/M p = 0.8905). Because 

the M/W host/donor group uniquely exhibited GID behavior, the additional groups (i.e., 

M/M, W/W, W/M) were consolidated to compare to the M/W group (Figure 3.4g; GID+ 

group vs. GID- group). No significant differences were observed when reported in this 

manner (GID+ M/W vs. 3 other GID- host/donor groups p = 0.4587). Further, to 

determine whether a correlation still exists between VGLUT2 expression in Met/Met 

hosts with WT grafts and GID behavior, a Spearman correlation was performed (Figure 

3.4h). In this study, the number of VGLUT2 protein expressed within the grafted DA 

neurons was not significantly correlated with total GID severity at 10 weeks post-

212 

 
engraftment (Figure 3.4h; r = 0.3571, p = 0.4444). Despite no longer being statistically 

correlated, a positive trend remains. As the number of VGLUT2 protein expression 

increased, the total GID severity score also increased. As presented below, additional 

evidence suggests that VGLUT2 expression in grafted DA neurons has a complex 

relationship to GID.  

a) 

b) 

c) 

Figure 3.4: Impact of host/donor genotype on development of GID behavior and 
association with VGLUT2 expression. 
(a) Total and (b) peak amphetamine-induced GID severity scores for all host/donor groups 
at week 10 post-engraftment. Statistics: Mean ± SEM. Non-parametric Kruskal-Wallis with 
Dunn’s multiple comparisons, p = 0.0325 M/W vs. sham-graft (total) and p = 0.0071 M/W 
vs.  sham-graft  (peak).  (c)  Percent  incidence  of  total (≥  4) and peak  (≥ 2)  GID severity 
score  in  all  host/donor  groups  at  10  weeks  post-engraftment.  Percentages  are  listed 
above  each  bar.  (d)  Time  course  of  amphetamine-mediated  GID  behavior  in  each 
host/donor at  week 10  post-engraftment at 20,  70-,  120-,  170-, and  220-minutes post-
amphetamine administration. Statistics: Non-parametric Kruskal-Wallis test with Dunn’s  
multiple  comparisons  at  each  time  point  post-amphetamine  injection.  70  minutes:  p  = 
0.0071 M/W vs. sham-graft. (e) Fluorescent image and Imaris 3D reconstruction of DA  

213 

 
 
Figure 3.4 (cont’d)  
(TH+) neurons  positive  for VGLUT2  protein co-expression.  Scale bar =  5  µm.  (f) Total 
quantification of the number of VGLUT2 protein co-localized in TH+ grafted DA neurons 
normalized to the surface volume (µm3) of the graft. Statistics: Mean ± SEM. One-way 
ANOVA with Tukey’s multiple comparisons, p ≥ 0.6513 for all host/donor groups. (g) Total 
quantification of VGLUT2 co-localization inside TH+ grafted DA neurons with the 3 GID- 
host/donor groups combined (i.e., M/M, W/W, W/M host/donor). Statistics: Mean ± SEM. 
Unpaired two-tailed t-test, not significant.  

214 

 d)  
 
 
 
 
 
 
 
Figure 3.4 (cont’d) 
(h) Spearman correlation comparing the total quantity of VGLUT2 co-localized inside TH+ 
neurons  and  total  amphetamine-mediated  GID  severity  scores  at  10  weeks  post-
engraftment. No significance. Abbreviations: GID = graft-induced dyskinesia, VGLUT2 = 
vesicular glutamate transporter 2, TH = tyrosine hydroxylase, ns = not significant, 3OR = 
3 other groups combined (i.e., M/M, W/W, W/M host/donor).  

Homozygous rs6265 (Met/Met) parkinsonian rats engrafted with WT DA neurons 

express lower BDNF receptor transcript ratios (TrkB to p75NTR) 

The two predominant receptors that BDNF binds to include tropomyosin receptor 

kinase B (TrkB) and the pan neurotrophin receptor (p75NTR) (Reichardt, 2006). Upon 

activation of the TrkB receptor, multiple signaling pathways involved in pro-survival and 

dendritic growth/branching are activated (Jaworski et al., 2005; Kumar et al., 2005). In 

contrast, when BDNF is bound to p75NTR, it is generally accepted that pro-apoptotic 

pathways are activated (Friedman, 2000; Meeker & Williams, 2015). In the literature, an 

imbalance between TrkB/ p75NTR proteins has been implicated in neurodegenerative 

rodent models of Huntington’s disease (HD) spine density (Brito et al., 2013; Suelves et 

al., 2019). Therefore, I hypothesized that an imbalance between TrkB and p75NTR 

transcript expression exists, with a prominent upregulation in p75NTR mRNA, and that 

215 

 
 
 
 
this imbalance is correlated with GID in Met/Met parkinsonian rats engrafted with WT 

DA neurons.  

The average quantity of the TrkB mRNA transcripts per cell in the striatum (TH+ 

and non-TH+ cells) was significantly increased in the Met/Met hosts engrafted with 

Met/Met DA neurons, however, only on the intact side (Figure 3.5b; M/W vs, M/M p = 

0.0481; M/M vs. W/M p = 0.0424; M/M vs. W/W p = 0.0383). On the grafted side, TrkB 

transcript expression was normalized in the presence of all DA neuron grafts (i.e., not 

significantly different between DA-grafted groups). Interestingly, p75NTR transcript 

expression was only found to be significantly upregulated between the M/M and W/W 

host/donor groups within the grafted DA neurons (Figure 3.5c; M/M vs. W/W graft p = 

0.0399). Further, slightly increased expression of p75NTR transcripts was present outside 

the DA graft (i.e., in TH- cells located dorsolateral from the graft), albeit this was not 

found to be statistically significant (data not shown).  

Since the imbalance of p75NTR/TrkB receptor expression has specifically been 

implicated in neurodegenerative diseases, we also reported the ratio of TrkB to p75NTR 

mRNA within the grafted TH+ neurons of each host/donor combination. When analyzed 

separately, no significant differences exist between groups (Figure 3.5d). However, 

when the three GID- groups are combined and compared to the GID+ M/W host/donor 

group, there is a notable decrease in the TrkB:p75NTR mRNA ratio in the grafted TH+ 

neurons (Figure 3.5f; M/W vs. M/M, W/M, W/W p = 0.0472), indicative of a relative 

increase in p75NTR receptors. While this was not significantly correlated with GID 

behavior at week 10 post-engraftment (Figure 3.5e; M/W r = -0.4058, p = 0.4333), a 

negative trend is apparent between the TrkB:p75NTR mRNA ratio and GID behavior 

216 

 
where a lower ratio was associated with a higher GID score. Moreover, total p75NTR 

mRNA expression inside grafted TH+ neurons was higher in the GID+ M/W host/donor 

group compared to the three other GID- host/donor combinations, albeit not statistically 

significant (Figure 3.5h; M/W vs. M/M, W/M, W/W p = 0.2824). Total TrkB mRNA 

expression inside grafted TH+ neurons was not different between groups (Figure 3.5g). 

These results suggest there is a trend toward upregulation of p75NTR mRNA expression 

in the M/W host/donor group which could potentially be associated with GID behavior; 

however, additional analyses such as protein expression are warranted.  

a) 

b) 

c) 

Figure 3.5: Impact of host/donor genotype on TrkB and p75NTR BDNF receptor 
transcript expression in DA-grafted parkinsonian rats. 
(a) Confocal fluorescent image and Imaris 3D reconstruction of TrkB and p75
puncta inside DA (TH+) neurons. Scale bar = 10 µm. (b) Total quantity of TrkB mRNA  

NTR

 mRNA 

217 

 
 
 
Figure 3.5 (cont’d) 
transcripts per cell (TH+ and TH-) in the intact and grafted striatum of each host/donor 
combination.  Statistics:  Mean  ±  SEM.  Two-way  ANOVA  with  Tukey’s’  multiple 
comparisons, p = 0.0481 M/W vs. M/M, p = 0.0383 M/M vs. W/W, p = 0.0424 M/M vs. 
W/M  in  the  intact  striatum.  No  significance  was  found  in  the  grafted  striatum  between  
host/donor groups, p ≥ 0.1612 for all groups. (c) Total quantity of p75
 mRNA transcripts 
per cell (TH+ and TH-) in the intact and grafted striatum of each host/donor combination. 
Statistics: Mean ± SEM. Two-way ANOVA with Tukey’s’ multiple comparisons, p = 0.0399 
M/M vs. W/W in the grafted striatum, p ≥ 0.4991 in the intact striatum for all host/donor 
combinations.  (d)  Ratio  of  TrkB:p75
  mRNA  per  TH+  grafted  DA  neuron.  Statistics: 
Mean  ±  SEM.  One-way ANOVA  with  Tukey’s  multiple  comparisons,  p  ≥  0.4189  for  all 
host/donor groups. (e) Ratio of TrkB:p75
 mRNA per TH+ grafted DA neuron with the 3 
GID- host/donor groups combined (i.e., M/M, W/W, W/M host/donor). Statistics: Mean ± 
SEM. Unpaired two-tailed t-test, p = 0.0472 M/W vs. 3OR. (f) Correlation of the ratio of 
TrkB:p75
  mRNA  per  TH+  grafted  DA  neuron  and  GID  score  at  10  weeks  post-
engraftment  in  M/W host/donors and  3OR  combined.  Statistics:  Spearman  correlation, 
not significant.  

NTR

NTR

NTR

NTR

d) 

e) 

218 

 
 
 
 
 
 
 
 
Figure 3.5 (cont’d) 
(g)  Total TrkB  mRNA  transcripts  and  (h)  p75
  alone  inside TH+  grafted  DA  neurons 
between  M/W  host/donor  and  3  other  host/donor  groups.  Statistics:  Mean  ±  SEM. 
Unpaired two-tailed t-tests, no significance. Abbreviations: TrkB = tropomyosin receptor 
kinase  B,  p75
  =  pan  neurotrophin  receptor,  TH  =  tyrosine  hydroxylase,  mRNA  = 
messenger ribonucleic acid, 3OR = 3 other host/donor groups combined.  

NTR

NTR

Aberrant GID behavior in homozygous rs6265 (Met/Met) parkinsonian recipients 

of WT DA grafts is associated with excess DA release 

Excess DA release is one of the mechanisms that has been postulated as an 

underlying cause of GID behavior (Politis, 2010b; Politis et al., 2011; Steece-Collier et 

al., 2012). To gain initial insight into this possible mechanism, I indirectly examined DA 

release in our host/donor combinations using immunohistochemical postmortem 

expression of the DAT protein. DAT is a transmembrane receptor that clears DA from 

the extracellular space following its release into the synapse. In order to clear increased 

concentrations of DA from the synapse, a compensatory upregulation of DAT is required 

(Lohr et al., 2017; Zhu & Reith, 2008). Thus, an increase in DAT expression is a 

surrogate marker indicative of an increase in DA release.  

219 

 
 
 
 
Although not statistically significant when examined as separate host/donor 

combination groups (Figure 3.6b), when combined by GID status, Met/Met hosts 

engrafted with WT DA neurons demonstrated a significant increase in DAT expression 

(i.e., DAT sum intensity/um2) in comparison to the GID- M/M, W/M, and W/W host/donor 

groups (Figure 3.6c; M/W vs. M/M, W/M, W/W p = 0.0085), suggestive of an increase in 

DA release in the GID+ group. Fluorescent intensity in these postmortem analyses is 

equivalent to DAT protein expression since the fluorescent staining pattern of DAT is 

ubiquitous and fills the entire neuron. Although exhibiting increased expression, DAT 

intensity in the M/W host/donor animals was not statistically correlated with total GID 

severity at the conclusion of the study (i.e., 10 weeks post-engraftment, Figure 3.6d), 

suggesting that, in these animals, while enhanced DA release may exist, it alone may 

not be sufficient for GID induction.  

220 

 
Figure 3.6: Impact of host/donor genotype on DAT expression in DA-grated 
parkinsonian rats. 
(a) Representative confocal fluorescent micrograph of depicting staining patterns of the 
dopamine transporter (DAT) and TH in the grafted parkinsonian striatum (cyan = DAT, red 
= TH). Scale bar = 300 µm; 50 µm for the inset image.  (b) DAT expression/fluorescent 
intensity  quantification  in  grafted  DA  neurons.  Data  are  expressed  as  the  sum  DAT 
intensity  per  DAT  surface  area  (µm2).  Statistics:  Mean  ±  SEM.  One-way ANOVA  with 
Tukey’s  multiple  comparisons,  p  ≥  0.2313  in  all  host/donor  groups.  (c)  DAT 
expression/fluorescent  intensity  quantification  in  grafted  DA  neurons,  demonstrated 
between  the  M/W  host/donor  group  and  the  other  host/donor  combinations  combined. 
Statistics:  Mean  ±  SEM.  Unpaired  two-tailed  t-test,  p  =  0.0085.  (d)  Non-parametric  
Spearman correlation between DAT sum intensity/DAT surface area (µm2) and GID score 
at  10  weeks  post-engraftment.  Statistics:  Spearman  correlation,  no  significance. 
Abbreviations:  DAT  =  dopamine  transporter,  TH  =  tyrosine  hydroxylase,  GID  =  graft-
induced dyskinesia.  

221 

 
 
GID behavior in homozygous rs6265 (Met/Met) parkinsonian rats engrafted with 

WT DA neurons is not correlated to immune marker expression in the 

parkinsonian striatum  

Another mechanism that has been speculated to underlie GID induction is 

increased activation of the immune system as detailed in Chapter 1 (Freed et al., 2001; 

Olanow et al., 2003; Soderstrom et al., 2008; Steece-Collier et al., 2012). Since existing 

evidence points to a promising influential role of the immune system in GID induction 

(Freed et al., 2001; Hagell et al., 2002; Olanow et al., 2003; Soderstrom et al., 2008), I 

investigated the expression of two common immune markers including ionized calcium-

binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) to provide a 

cursory examination in this study.  

Microglial (Iba1) is an immune marker involved in generation and elimination of 

synaptic connections (Tremblay et al., 2011). In this study, Iba1 was used as an 

indication of inflammation and quantified in the striatum of all host/donor subjects. As a 

marker for astrocytes, which, upon immune activation, can release proinflammatory 

cytokines and chemokines (Giovannoni & Quintana, 2020), GFAP was analyzed as 

another indicator of inflammation. I hypothesized that Iba1 and GFAP expression would 

be increased in M/W host/donors, and that this would correlate to GID behavior. Iba1 

and GFAP expression were reported as Iba1 volume (µm3)/TH+ neuron and GFAP 

volume (µm3)/TH+ neuron, respectively.  

Contrary to this hypothesis, Iba1 expression per TH+ neuron was not significantly 

different between genotypic host/donor combinations, even when M/M, W/M, and W/W 

(GID-) groups are combined (Figure 3.7bc; M/W vs. M/M, W/M, W/W p = 0.2894). 

222 

 
Moreover, Iba1 expression was also not correlated to GID behavior (Figure 3.7d; M/W r 

= 0.2674, p = 0.2834). Likewise, the same outcome was demonstrated for GFAP 

expression (Figure 3.7ef; M/W vs. M/M, W/M, W/W p = 0.5260). While there is a slight 

positive trend between GFAP expression and total GID severity 10 weeks post-

engraftment, statistical significance was not apparent (Figure 3.7g; M/W r = 0.1786, p = 

0.7131).  

Figure 3.7: Impact of host/donor genotype on immune marker (Iba1 and GFAP) 
expression in parkinsonian rats. 
(a)  Representative  confocal  fluorescent  micrograph  illustrating  the  presence  of  Iba1+ 
(red) and GFAP+ (cyan) cells in the grafted parkinsonian striatum. Scale bar = 20 µm. (b) 
Quantity of Iba1+ cells normalized to the number of TH+ grafted DA neurons, expressed 
as Iba1 volume (um3)/TH neuron in each host/donor combination. Statistics: Mean ± SEM. 
One-way ANOVA with Tukey’s multiple comparisons, p ≥ 0.3433 in all host/donor groups. 
(c) Quantity of Iba1 volume (um3)/TH neuron between the M/W host/donor group and the  
3 other host/donor combinations combined. Statistics: Mean ± SEM. Unpaired two-tailed 
t-test, no significance (p = 0.2894). (d) Spearman correlation between quantity of Iba1  

223 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.7 (cont’d) 
volume (um3)/TH neuron and total GID severity at 10 weeks post-engraftment. Statistics: 
Spearman  correlation,  no  significance.  (e)  Quantity  of  GFAP+  cells  normalized  to  the 
number of TH+ grafted DA neurons, expressed as Iba1 volume (um3)/TH neuron in each 
host/donor combination. Statistics: Mean ± SEM. One-way ANOVA with Tukey’s multiple 
comparisons, p ≥ 0.5161 in all host/donor groups. (f) Quantity of GFAP volume (um3)/TH 
neuron  between  the  M/W  host/donor  group  and  the  3  other  host/donor  combinations 
combined.  Statistics:  Mean  ±  SEM.  Unpaired  two-tailed  t-test,  no  significance  (p  = 
0.5260). (g) Spearman correlation between quantity of GFAP volume (um3)/TH neuron 
and total GID severity at 10 weeks post-engraftment. Statistics: Spearman correlation, no 
significance. Abbreviations: Iba1 = Ionized calcium binding adaptor molecule 1, GFAP = 
glial  fibrillary  acidic  protein,  3  other  host/donor  groups  combined,  GID  =  graft-induced 
dyskinesia.  

224 

 
 
 
 
 
DISCUSSION 

The primary objective of DA neuron transplantation therapy is to provide a safe 

and effective additional or alternative treatment option to the current therapies (e.g., DA 

replacement therapy) used to treat PD. However, while substantial progress has been 

made in neural grafting over the past two decades (Barker et al., 2024), mechanisms 

underlying heterogeneity in clinical responsiveness remains unknown with GID 

remaining a significant, aberrant side effect. Despite reinvigorated interest, with several 

clinical trials planned or ongoing ((Barker et al., 2019); clinical trial identifier examples 

NCT04802733, NCT01898390, NCT03309514, NCT03119636, NCT04146519), the 

question remains whether we understand the mechanisms underlying regenerative cell 

therapy enough for its safe incorporation into clinical practice. Until we can achieve 

optimal benefit while preventing side effect liability, neural transplantation will not be 

considered a viable, effective alternative therapeutic option for individuals with PD.  

As discussed previously, our laboratory became interested in the common human 

SNP, rs6265, as a potential risk factor underlying the variability of clinical outcomes in 

DA neuron transplantation. Using a CRISPR knock-in parkinsonian rat model of the 

rs6265 SNP, we have demonstrated that homozygous rs6265 (i.e., Met/Met) 

parkinsonian rats engrafted with WT DA neurons exhibited enhanced therapeutic 

efficacy evidenced by earlier and more robust amelioration of LID behavior post-

engraftment in comparison to grafted WT subjects. Moreover, a paradoxical 

enhancement of graft-derived neurite outgrowth was reported in these Met/Met animals 

(Mercado et al., 2021). This finding was contrary to our hypothesis since BDNF normally 

promotes dendritic spine and synapse formation within the striatum (Gonzalez et al., 

225 

 
2016; Kowiański et al., 2018; Park & Poo, 2013; Sasi et al., 2017; Zagrebelsky et al., 

2020) and increases graft-derived innervation in parkinsonian rats (Yurek, 1998; Yurek 

et al., 1996). Because the Met/Met genotype has decreased activity-dependent release 

of BDNF, we had theorized that grafted Met/Met parkinsonian rats would demonstrate 

diminished neurite outgrowth instead.  

While seemingly a paradoxical phenomenon, research groups of other disease 

models have also highlighted a benefit of the rs6265 Met allele. For example, Met-allele 

carriers expressed enhanced recovery and axon regeneration following TBI in combat 

veterans (Barbey et al., 2014; Finan et al., 2018; Krueger et al., 2011). Remarkably, 

Met-allele carriers with MS (Zivadinov et al., 2007) or late-stage AD (Voineskos et al., 

2011) have reported a reduction in cognitive decline compared to WT patients. Other 

preclinical studies in rodents have also reported similar findings (McGregor et al., 2019; 

McGregor & English, 2019). Collectively, this evidence supports the notion that the 

rs6265 SNP may confer protective, or neuroregenerative, effects in disease and likely 

has an evolutionary benefit (Di Pino et al., 2016).  

Intriguingly, the BDNF prodomain/pro-peptide has been recently speculated as 

being responsible for the potential neuroregenerative effect of the Met allele. Because 

the rs6265 SNP is found within the BDNF prodomain/pro-peptide region, and because 

the pro-peptide has recently been discovered to function as an independent ligand 

similar to that of proBDNF and mature BDNF (Anastasia et al., 2013), it is reasonable to 

suggest that the Met BDNF pro-peptide could have an unexpected benefit of growth-

enhancing properties in neural grafting. While some evidence shows differential 

functions of the Val- and Met-type BDNF pro-peptide (e.g., (Anastasia et al., 2013), 

226 

 
findings are limited to the hippocampus, and further research will be required to fully 

elucidate their function in the grafted parkinsonian striatum (see (Szarowicz et al., 2022) 

for a comprehensive discussion of the BDNF pro-peptide).  

With our current study, I endeavored to investigate rs6265 in both host and donor 

neurons on functional outcomes of neural transplantation to understand fully the impact 

of the Met allele in the host and donor. Due to the high prevalence of rs6265 in the 

general population (i.e., 20%) (Petryshen et al., 2010; Tsai, 2018), the odds of a PD 

patient receiving a graft containing a Met allele is inevitable. Studies which precede this 

(Mercado et al., 2021, 2024) only engrafted WT DA neurons, and to our knowledge, this 

is the first experiment of its kind to examine rs6265 in both host and donor in a 

parkinsonian rat model. Thus, we engrafted WT and Met/Met parkinsonian host rats with 

either WT or Met/Met donor neurons, generating six different host/donor combinations 

including sham-grafted subjects. My goal was to determine the optimal host/donor 

combination that retained graft-derived functional benefit but had diminished side effect 

liability (i.e., GID). 

As the primary behavioral readout of graft function, amelioration of LID was 

employed. In our previous study, Met/Met parkinsonian host rats engrafted with WT DA 

neurons demonstrated earlier and more robust amelioration of LID behavior over the 

entire 10-week time course compared to their WT counterparts (Mercado et al., 2021). 

In the current study, I hypothesized that host and/or donors with the Met/Met genotype 

would retain behavioral benefit and exhibit earlier amelioration of LID, based on our 

previous research and other evidence of the Met-allele benefit as discussed above 

(Barbey et al., 2014; Finan et al., 2018; Krueger et al., 2011; McGregor et al., 2019; 

227 

 
McGregor & English, 2019). In line with this hypothesis, the Met/Met genotype permitted 

a slightly earlier functional recovery (i.e., lower LID behavior), regardless of host or 

donor, when compared to WT hosts engrafted with WT DA neurons. All grafted groups 

in which the Met/Met genotype was present demonstrated a significant functional 

benefit (i.e., decrease in LID) starting at week four post-engraftment, whereas the 

WT/WT (host/donor) group did not demonstrate statistically significant recovery until 

week eight post-engraftment. Interestingly, at the completion of the study (10 week post-

engraftment), the WT/WT host/donor rats lost statistically significant functional benefit 

while the LID behavior of the other three Met/Met host/donor groups remained 

significantly lower compared to sham-grafted animals.  

I also employed amphetamine-mediated rotational behavior as a secondary 

readout of graft function (Collier et al., 1999, 2015; Dunnett & Torres, 2011; Soderstrom 

et al., 2008). Not only does the Met/Met genotype permit behavioral recovery through 

amelioration of LID behavior, these animals also exhibited a lower number of ipsilateral 

amphetamine-induced rotations per minute at 10 weeks post-engraftment. For example, 

similar to LID analysis, the groups containing a Met/Met genotype (i.e., M/W, M/M, W/M 

host/donors) collectively demonstrated a statistically significant reduction in 

amphetamine-mediated rotations compared to sham-grafted rats at 10 weeks post-

engraftment. This difference was not apparent in the WT/WT host/donor subjects. 

Results collected from LID ratings and amphetamine rotational analysis further confirm 

that the Met-allele indeed retains functional benefit.  

Although a significant increase in neurite outgrowth was prevalent in Met/Met 

hosts engrafted with DA neurons in our previous study (Mercado et al., 2021), this 

228 

 
difference was not apparent in this current experiment 10 weeks post-engraftment. 

Estimated total grafted DA neurons and graft volume (µm3) were the same across 

host/donor combinations. Likewise, neurite density measurements per grafted DA 

neuron were not different among host/donor combinations, either proximal or distal 

distances from the graft. Because a notable difference is no longer detected, it is 

possible that the Met-allele-containing groups showed earlier enhanced outgrowth 

supported by functional data that was lost to detection over the 10 week time span, or it 

can be speculated that these animals have similar neurite densities because of more 

advanced host age: these rats are slightly older than those in our previous study 

(Mercado et al., 2021) by 2-3 months. Notably, in our middle-aged cohort (Mercado et 

al., 2024), enhanced neurite outgrowth between Met/Met and WT hosts was also no 

longer evident at 10 weeks post-engraftment. Therefore, the modest increase in age 

could have had an effect on neurite outgrowth in the animals of this current study, 

reaching a threshold and no longer presenting as an enhancement in the Met/Met 

genotype. As this is only speculation, an age-matched experiment would be necessary 

in the future.  

Because our overarching hypothesis was based on the idea that a decrease in 

activity-dependent BDNF release (i.e., rs6265) could underlie the variability in clinical 

responsiveness to neural grafting (i.e., GID induction), we postulated that Met/Met hosts 

engrafted with Met/Met donor neurons would display the greatest severity of GID 

behavior since both host and donor have a deficit in released BDNF. Unexpectedly, 

however, Met/Met hosts engrafted with WT DA neurons were the only group to develop 

significant GIDs compared to all other host/donor combinations. Although unexpected, 

229 

 
this finding does corroborate our findings in (Mercado et al., 2021), which demonstrated 

that Met/Met hosts engrafted with WT DA neurons uniquely developed GID compared to 

WT hosts. Nevertheless, a possible explanation as to why GID only develop in the M/W 

host/donor animals remains unknown. 

An earlier publication by our group ultrastructurally demonstrated that grafted DA 

neurons make asymmetric, atypical (presumed glutamatergic) synapses onto host 

MSNs in the striatum, and that the presence of these synapses positively correlated to 

increased GID behavior (Soderstrom et al., 2008). Consistent with these findings, 

Mercado and colleagues later reported that GID behavior in Met/Met host rats was 

strongly correlated to expression of VGLUT2 protein in the grafted DA neurons 

(Mercado et al., 2021). This is indicative that the grafts are maintaining an immature 

phenotype following transplantation, forming glutamatergic (asymmetric) synaptic 

connections onto striatal MSNs (El Mestikawy et al., 2011). Therefore, in my study, I 

also investigated the expression, and potential correlation, between VGLUT2 and GID 

behavior in the M/W host/donor group to ascertain whether this phenomenon was 

preserved. No longer was a statistical correlation found between VGLUT2 expression 

and GID behavior in these animals; however, a similar trend still existed. Due to the 

expression of VGLUT2 in the grafted neurons, it is still apparent that these grafts are 

maintaining an immature phenotype, yet this phenotype alone may not be sufficient to 

underlie GID behavior.  

As discussed above, it has been reported that an imbalance between TrkB/ 

p75NTR proteins is implicated in neurodegenerative rodent models such as HD (e.g., 

(Suelves et al., 2019)). Specifically, Suelves and colleagues investigated the impact of 

230 

 
the p75NTR/TrkB imbalance on motor behavior and striatal neuropathology in a HD 

mouse model (Suelves et al., 2019). Their results demonstrated increased levels of 

p75NTR in the striatum of HD mice associated with the manifestation of motor 

abnormalities and a decrease in dendritic spine density. Once p75NTR levels were 

genetically normalized, dendritic spine density was rescued, and motor deficits were 

delayed (Suelves et al., 2019). Other studies have similarly confirmed these findings 

(see (Brito et al., 2013; Zagrebelsky et al., 2020; Zuccato et al., 2008)). Of note, p75NTR 

can also play a critical role in glutamate synaptogenesis where its activation can 

influence synapse development and glutamate release (Numakawa et al., 2003; Wang 

et al., 2022).  

Because of the aberrant nature of grafted DA synapses onto MSN dendrites in 

the presence of decreased spine densities in the parkinsonian striatum (e.g., 

(Soderstrom et al., 2008)), I hypothesized that an imbalance between TrkB and p75NTR 

transcript and receptor expression, with a propensity toward p75NTR upregulation, would 

correlate with GID behavioral development in Met/Met parkinsonian rats engrafted with 

WT DA neurons. Since upregulation of p75NTR has been associated with a decrease in 

dendritic spine density (Reichardt, 2006; Zagrebelsky et al., 2005), it is reasonable to 

suggest that, if the M/W host/donor group presents with an increase in p75NTR, they may 

also demonstrate decreased spine density, which could impact synaptic circuitry 

between the host MSNs and the grafted DA neurons, ultimately leading to GID 

development. Moreover, if an upregulation of p75NTR is found within the grafted DA 

neurons, it could suggest an activation of glutamate release from the DA neurons onto 

host MSNs.  

231 

 
Expectedly, in the intact striatum, there was an upregulation of TrkB mRNA 

expression in the Met/Met parkinsonian hosts engrafted with Met/Met DA neurons. 

Mercado and colleagues previously noted a similar finding where the intact side of the 

Met/Met parkinsonian hosts demonstrated an upregulation of TrkB mRNA compared to 

WT hosts (Mercado et al., 2021). Biologically, an upregulation of TrkB is expected in the 

homozygous rs6265 Met/Met genotype as there is a decrease in BDNF in the brain 

microenvironment (Egan et al., 2003). Additionally, we report no differences in TrkB 

mRNA expression in grafted DA neurons of all host/donor combinations, which is also in 

confirmation of the findings reported by Mercado and colleagues where Met/Met and 

WT DA-grafted parkinsonian animals expressed similar levels of TrkB mRNA (Mercado 

et al., 2021). Likewise, expression of p75NTR mRNA was not statistically different 

between groups in the intact striatum or grafted DA neurons; however, there appeared 

to be a slight increase in p75NTR transcripts inside the graft in M/W host/donor animals. 

Variability within this group was substantial and likely accounts for the lack of statistical 

significance of this increase.  

Most importantly, when reported as a ratio (i.e., TrkB:p75NTR) within TH+ neurons, 

GID+ M/W host/donor rats exhibit a significantly lower ratio compared to the three other 

GID- host/donor groups combined, suggesting that there are more p75NTR transcripts 

per grafted DA neuron than TrkB transcripts in this host/donor combination. Based on 

the mechanism of action known for p75NTR (Friedman, 2000; Teng et al., 2005; Woo et 

al., 2005), we can infer that an increased presence of p75NTR may prevent proper 

formation of typical, symmetric DA neuron circuitry, potentially causing GID. Additionally, 

p75NTR activation on the grafted DA neurons could lead to glutamate release onto host 

232 

 
MSNs, also potentially resulting in GID (see DA/glutamate co-transmission below). 

Nevertheless, total GID severity at 10 weeks post-engraftment was not statistically 

correlated with the TrkB:p75NTR ratio, indicating that this imbalance may still be 

necessary but not a sufficient sole contributor of GID. Since the quantity of mRNA 

transcripts does not always coincide 1:1 to protein expression, further studies that 

examine both TrkB and p75NTR protein levels are necessary to definitively determine the 

role of these receptors in this animal model. Furthermore, investigating activation state 

of BDNF receptors (e.g., phosphorylated) could provide additional insight into any 

potential changes in activity that could correlate to the expression of aberrant GID 

behaviors. 

A consistent mechanism that has been posited as underlying GID behavior is 

uneven and/or excess DA release. Specifically, excess DA was first reported in PD 

patients who developed aberrant GID in the first double-blind clinical trials (for review 

(Piccini et al., 1999; Politis, 2010a; Politis et al., 2011)). Supporting the role of DA and 

its receptors in GID, Shin and colleagues demonstrated confirmatory evidence in DA-

grafted parkinsonian rats, demonstrating that pharmacological blockade of D2 (i.e., 

eticlopride, buspirone) and D1 (i.e., SCH23390) receptors resulted in almost complete 

amelioration of GID behavior. Moreover, although buspirone is also primarily considered 

a partial 5-HT receptor agonist, blockade of D2 was found to be independent from 

activation of 5-HT because its effect was not prevented by a 5-HT antagonist (Shin et 

al., 2012). This preclinical model ultimately supports the action of buspirone in D2 

receptor antagonism and points to a promising role of DA release in GID behavior.  

233 

 
Therefore, I analyzed immunohistochemical expression of DAT as a surrogate 

marker of DA release in postmortem tissue. Confirming my hypothesis, GID+ M/W 

host/donor animals demonstrated a significant increase in DAT expression (DAT sum 

intensity/µm3) compared to the three other GID- host/donor groups (i.e., M/M, W/M, 

W/W), indirectly indicating that more DA is being released in the M/W host/donor 

parkinsonian rats. Because Mercado and colleagues illustrated that there was an 

upregulation of Drd2 mRNA (DA D2 receptor) in Met/Met hosts (Mercado et al., 2024) 

compared to WT hosts, it is further reasonable to speculate that increased DA release 

from the WT graft in an environment with (presumably) upregulated D2 receptors could 

increase activation of host MSNs, subsequently permitting GID behavior. Despite this 

logical postulation, there was no statistical correlation between GID severity and DAT 

expression, again suggesting that, while an increase in DAT expression may be 

necessary, DAT expression alone may not be sufficient to induce GID behavior. 

Additional studies that examine direct release of DA will be necessary.   

Immune system activation is another promising mechanism that could potentially 

underlie GID behavior. As stated above, our group has previously shown that DA-

grafted parkinsonian rats exhibited increased GID severity following immune challenge 

(Soderstrom et al., 2008). In grafted patients with PD, GID developed upon cessation of 

immune suppression (Hagell & Cenci, 2005; Olanow et al., 2003). Therefore, I 

investigated two common markers of the immune system including Iba1 (microglia) and 

GFAP (astrocytes) to provide cursory insight into whether the immune system impacted 

GID expression in my studies. However, no obvious differences were found between 

host/donor combinations in either Iba1 or GFAP expression. Moreover, no correlation 

234 

 
was exhibited between Iba1 or GFAP expression and total GID severity at the 

conclusion of the study. Although no correlation was evident with these specific 

markers, a role for immune activation should not be excluded as a potential GID 

mechanism based on historical data. Here, only pan markers for microglia and 

astrocytes that stain nearly all Iba1+ and GFAP+ cells in the brain were employed. 

Markers for activated immune factors such as major histocompatibility complex 2 (MHC-

II) should be utilized for greater specificity in the future. Additionally, directly assessing 

the connection between immune suppression and GID induction in association with the 

rs6265 SNP in this parkinsonian rat model is warranted.  

It is highlighted here that graft-derived functional benefit of the rs6265 (Met/Met) 

genotype is retained in parkinsonian rats whether it is found in the host or donor, and 

that the Met/Met hosts engrafted with WT DA neurons remain the only host/donor 

combination to develop aberrant GIDs. While we are aware that advances in the clinical 

grafting field have been made, with several clinical trials planned or ongoing ((Barker et 

al., 2019); clinical trial identifier examples NCT04802733, NCT01898390, 

NCT03309514, NCT03119636, NCT04146519), we recognize that a gap in our 

understanding regarding the underlying mechanism of GID still exists. In our continuing 

investigation of GID, we have established a probable role of excess DA release in this 

aberrant behavior—a finding that has been also supported in grafted PD patients who 

received buspirone (a drug with DA antagonist properties) that successfully reduced 

their GID (Politis, 2010a; Politis et al., 2011; Steece-Collier et al., 2012). The exact 

mechanism(s) that result in the association between GID and DA release warrants 

further investigation in preclinical models and clinical trials. Moreover, because it is not 

235 

 
common practice in clinical grafting trials, our strong cumulative data suggests that both 

participants and donor neurons are genotyped for the rs6265 SNP prior to 

transplantation. Once the field can harness the benefit, while preventing the detriment, 

of the rs6265 SNP, regenerative cell therapy has the potential to be a fully optimized 

therapeutic option to treat not only PD, but also other neurodegenerative and 

neurological disorders.  

236 

 
 
 
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CHAPTER 4: EXOGENOUS BDNF TREATMENT EXACERBATES GRAFT-INDUCED 
DYSKINESIA IN HOMOZYGOUS rs6265 (MET/MET) PARKINSONIAN RATS 

246 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ABSTRACT 

Dopamine (DA) neuron transplantation remains a promising therapeutic 

approach to restore lost DA in the parkinsonian striatum; however, a significant side 

effect of is graft-induced dyskinesia (GID). While several theories of GID have been 

posited, its underlying mechanisms remain unclear and controversial. Our 

investigations, aimed at understanding potential genetic contributions to GID, have 

focused on a common single nucleotide polymorphism (SNP), rs6265, found in the gene 

for brain-derived neurotrophic factor (BDNF), which results in decreased BDNF release. 

Using a CRISPR knock-in rat model, we first reported that parkinsonian rats 

homozygous for rs6265 (aka Met/Met) engrafted with wild-type (WT) primary DA 

neurons uniquely developed GID compared to their WT counterparts. Because rs6265 

causes decreased BDNF release, we hypothesized that “replenishing” BDNF would 

ameliorate GID behavior. To evaluate this, exogenous intracerebral BDNF was infused 

into parkinsonian Met/Met rats engrafted with WT DA neurons using osmotic 

minipumps. Unexpectedly, BDNF infusion exacerbated GID in grafted Met/Met animals 

compared to vehicle-infused controls, and evidence suggests that dysregulated 

DA/glutamate co-transmission and/or excess DA release contributes to GID expression. 

While our findings are supported by clinical data, they reveal novel mechanisms that are 

related to an individual’s genetic profile that may be important to consider as cell 

transplantation therapies advance in ongoing clinical trials.  

247 

 
 
INTRODUCTION 

PD is a complex, heterogeneous neurodegenerative disorder that affects over 

9.3 million people worldwide (Espay et al., 2017; Maserejian et al., 2020; Schalkamp et 

al., 2022). While pharmacological interventions traditionally used to treat PD (e.g., 

levodopa) alleviate a majority of motor symptoms, there is significant heterogeneity in 

clinical responsiveness (Fischer et al., 2018, 2020; Hauser et al., 2009; Sortwell et al., 

2022), and most individuals eventually experience waning efficacy and side effect 

development (e.g., levodopa-induced dyskinesia (LID)) as their disease progresses 

(Hauser et al., 2009). Based on the unmet need in clinical management of PD, 

additional/alternative therapies continue to be investigated (e.g., (Barker et al., 2024)). A 

promising regenerative medicine alternative involves DA neuron transplantation aimed 

at restoring DA terminals within the striatum to replace those that die off in PD. The 

transplantation method that has had most clinical success is grafting primary embryonic 

ventral mesencephalic (eVM) DA neurons into the caudate/putamen, demonstrating 

clear efficacy in a subpopulation of recipients with PD (Olanow et al., 2009; Steece-

Collier et al., 2012; Stoker et al., 2017). Despite distinct, yet heterogenous, success, 

clinical trials have also demonstrated heterogeneity in side effect development. 

Specifically, a subpopulation of patients exhibited a aberrant side effect known as GID in 

response to receiving DA neuron grafts (Freed et al., 2001; Hagell et al., 2002; Olanow 

et al., 2003). It was the occurrence of GID behavior that led to a worldwide moratorium 

on all clinical grafting trials in 2003 (Barker et al., 2019; Collier et al., 2019; Parmar et 

al., 2020; Stoker & Barker, 2020). After decades of rigorous preclinical research and 

retrospective analyses of clinical trials, several clinical grafting trials are planned or 

248 

 
ongoing (example clinical trial identifier examples NCT04802733, NCT01898390, 

NCT03309514, NCT03119636, NCT04146519), yet the underlying mechanism of GID 

behavior remains unclear and controversial.  

GID are abnormal involuntary movements that, to date, have been observed to 

manifest only in individuals who received primary DA grafts (for review (Steece-Collier 

et al., 2012)). Several proposed underlying mechanisms of GID have been posited. 

These include, but are not limited to, uneven DA reinnervation/excess DA release, 

donor cell source and preparation, presence of non-DA cells (e.g., serotonin neurons), 

age of recipient, pre-graft levodopa history, the immune response, and 

abnormal/asymmetric synaptic connections (Ma et al., 2002; Mercado et al., 2021, 

2024; Pagano et al., 2018; Soderstrom et al., 2008). Previously, our laboratory 

demonstrated that there was a significant association between GID behavior and the 

presence of excitatory asymmetric synaptic connections made by and onto grafted DA 

neurons in parkinsonian rats (Mercado et al., 2021; Soderstrom et al., 2008). Normally, 

mature DA neurons make en passant, symmetric appositions onto the dendritic spines 

of striatal medium spiny neurons (MSNs) (Gerfen & Surmeier, 2011; W. Shen et al., 

2016). However, the grafted neurons in the GID-expressing parkinsonian rats have 

been reported to make atypical asymmetric synapses directly onto the dendrite or onto 

the cell soma (Soderstrom et al., 2008). These atypical synaptic profiles have also been 

observed in PD patients engrafted with embryonic DA neurons (Kordower et al., 1996), 

suggestive of impaired and/or delayed maturation in which a DA-glutamate co-

transmission phenotype is common (El Mestikawy et al., 2011). 

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Given the necessity of BDNF for midbrain DA neuron maturation and synapse 

formation (Adachi et al., 2005; Baquet et al., 2005; Hyman et al., 1991; Yurek, 1998; 

Yurek et al., 1996), we began investigating the role of BDNF in GID behavior. We 

identified a common SNP, rs6265, found within the gene for BDNF which results in a 

decrease in activity-dependent BDNF release (Egan et al., 2003). The rs6265 SNP, also 

referred to as Val66Met, involves a valine to methionine substitution at codon 66 and 

occurs in approximately 20% of the general population (Petryshen et al., 2010; Tsai, 

2018). Both the heterozygous (Val/Met) and homozygous (Met/Met) genotype result in a 

significant dose-dependent decrease of activity-dependent release of BDNF by 

disrupting the packaging of BDNF into secretory vesicles (for review (Egan et al., 2003; 

Mercado et al., 2021)). Notably, rs6265 is not associated with PD incidence (Egan et al., 

2003; Mariani et al., 2015) but has been shown to reduce therapeutic efficacy of oral 

levodopa in PD patients (Fischer et al., 2020). Due to the considerable prevalence of 

rs6265 in the general population and the critical role of BDNF in promoting dendrite 

spine growth, formation of synapses in DA neurons, and maturation of DA neurons, I 

hypothesized that this genetic risk factor underlies the variability (i.e., GID behavior) in 

clinical response to DA neuron grafting in individuals with PD. We theorized that the 

decrease in BDNF release in the extracellular environment caused by the homozygous 

SNP (Met/Met) prevents proper graft maturation and that “replacing” the deficient BDNF 

would allow for graft maturation and proper integration into the host, ultimately 

ameliorating GID.  

Using a CRIPSR knock-in parkinsonian rat model of the homozygous rs6265 

SNP (Met/Met) developed by our colleagues Dr. Caryl Sortwell and Dr. Timothy Collier, 

250 

 
we recently demonstrated that only Met/Met rats engrafted with WT DA neurons 

uniquely exhibited induction of GID behavior compared to their WT counterparts 

engrafted also with WT DA neurons ((Mercado et al., 2021); see also Chapter 3)). In an 

attempt to mitigate GID behavior in an environment of decreased extracellular BDNF 

(i.e., rs6265 Met/Met), in this current study, we infused exogenous mature BDNF into 

the striatum of Met/Met host rats engrafted with WT DA neurons. We achieved 

exogenous BDNF administration with a subcutaneous osmotic minipump attached to a 

cannula that was placed directly above the grafted DA neurons in the parkinsonian 

striatum.  

We report here that, contrary to our hypothesis, exogenous BDNF infusion into 

DA-grafted homozygous Met/Met parkinsonian rats increased aberrant GID behavior 

compared to DA-grafted vehicle-infused controls. We also provide evidence that GID in 

these animals are correlated with indices of excess DA release demonstrated by an 

increase in DA transporter (DAT) expression and contralateral amphetamine-mediated 

rotational behavior. Importantly, these results corroborate findings in grafted PD patients 

where excess DA release was also found to be associated with GID (Piccini et al., 1999; 

Politis, 2010; Politis et al., 2011). Moreover, we provide evidence suggestive of an 

entirely novel mechanism associated with excess graft-derived DA signaling—a 

phenomenon known as vesicular synergy. Vesicular synergy, in this context, posits that 

the presence of vesicular glutamate transporter 2 (VGLUT2) on a vesicular monoamine 

transporter 2 (VMAT2)-positive synaptic vesicle within a DA neuron promotes increased 

DA loading into vesicles, resulting in excess DA release (El Mestikawy et al., 2011; 

Hnasko et al., 2010; H. Shen et al., 2021). Our supporting evidence illustrates high-

251 

 
resolution confocal imaging of (presumed) co-localized VMAT2 and VGLUT2 protein in 

striatal DA-grafted neurites, which is remarkably correlated with GID behavior. 

GIDs are a complex behavior in which my cumulative data suggest that several 

mechanisms appear to be necessary, but possibly not sufficient by themselves, to 

induce GID. Collectively, our research suggests that atypical DA/glutamate co-

transmission and/or excess DA release are promising factors underlying GID induction, 

influenced by genetic characteristics of the host and donor. However, additional 

research is necessary to directly confirm that excess DA release underlies GID and to 

fully understand GID pathogenesis. Our findings reinforce the notion that a personalized 

medicine approach will be imperative to optimize clinical outcomes of cell 

transplantation for individuals with PD. Once we understand the mechanisms of GID, 

we may be able to provide a solution to prevent its occurrence in this host/donor 

combination. 

252 

 
 
 
Experimental Animals 

METHODS 

Male Sprague-Dawley rats homozygous for rs6265 (Met/Met) (6-7 months at 

lesioning; 11-12 months at sacrifice) were obtained from our in-house colony derived 

from CRISPR knock-in rats carrying the valine to methionine polymorphism in the rat 

BDNF gene (Val68Met, Val/Met). Using CRISPR/Cas-mediated homologous 

recombination, these rats were generated by Cyagen Biosciences (Santa Clara, CA). In 

this study, only homozygous Met/Met rats were used based on findings from our 

previous experiments which demonstrated that Met/Met host rats engrafted with wild-

type (WT) DA neurons uniquely developed GID behavior (Mercado et al., 2021). Of 

note, the rat Val68Met SNP is equivalent to the human Val66Met SNP because rats 

have two additional threonine amino acids at positions 57 and 58. The BDNF gene in 

rats has approximately a 96.8% sequence homology with the human BDNF gene 

(BLAST queries: P23560 and P23363). The Michigan State University Institutional 

Animal Care and Use Committee approved all experimental procedures.  

Two rats were removed from experimental evaluation due to spontaneous death 

during or following neural transplantation surgery. Other animals (N=18) were excluded 

a priori (i.e., prior to grafting) because they failed to develop sufficient LID, as well as to 

keep the N of each group to approximately 10. A small number of (N=4) were excluded 

from postmortem analyses due to having too few surviving cells in the graft (<100) or 

misplaced grafts (e.g., cortically placed grafts). Final experimental cohorts were N=9 

BDNF (non-grafted), N=9 PBS (non-grafted), N=9 BDNF-infused (grafted), N=9 PBS-

infused (grafted).  

253 

 
Experimental Timeline 

Illustrated in Figure 4.1, rats were first unilaterally rendered parkinsonian via a 

stereotaxic injection of 6-hydroxydopamine (6-OHDA) in the SN and medial forebrain 

bundle (MFB). Lesion status was then confirmed two weeks later with amphetamine-

mediated rotational behavior as described in Chapter 3. Two weeks following, rats were 

primed with daily levodopa to induce significant, stable LID, which was our primary 

behavioral readout of graft function. After four weeks of priming with levodopa, rats 

received intrastriatal grafts of embryonic ventral mesencephalic (VM) DA neurons from 

wild-type (WT; Val/Val) rats or no grafts as the control. Immediately following grafting 

surgery, subcutaneous osmotic minipumps containing either mature BDNF (R&D 

Systems, Inc. Bio-Techne Corporation catalog # 11166-BD) or vehicle-control 

phosphate-buffered saline (PBS) were implanted under the skin. The minipumps were 

attached to a cannula that was placed directly above the grafted cells. Following grafting 

surgery, levodopa was withdrawn for one week and then reinitiated for the remainder of 

the study. Parkinsonian rats were evaluated for amelioration of LID behavior 10 weeks 

following engraftment. At five and 10 weeks post-engraftment, amphetamine-induced 

rotational behavior was assessed as a secondary measure of graft function. Lastly, as 

an indicator of graft dysfunction, GID were evaluated at five and 10 weeks following LID 

assessment.  

Nigrostriatal Lesioning with 6-OHDA 

Anesthetized (2% isoflurane, Sigma St. Louis, MO, USA) rats, after being placed 

in a stereotaxic frame, received two L of 6-OHDA (flow rate of 0.5 L/minute) to the 

SNpc (4.8 mm posterior, 2.0 mm lateral, 8.0 ventral relative to bregma) and the MFB 

254 

 
(4.3 mm posterior, 1.6 mm lateral, 8.4 mm ventral relative to bregma). After surgery 

completion, rats received intraperitoneal (i.p.) injections of 5 mg/kg carprofen (Rimadyl) 

as an analgesic. For histological postmortem confirmation of successful nigral lesions, 

medial terminal nucleus (MTN) DA cell enumeration methods were used (Gombash et 

al., 2014).  

Figure 4.1: Experimental Design and Timeline. 
(a) Experimental timeline of surgeries, behavioral evaluation, and drug administration. (b) 
Experimental schematic illustrating cell transplantation. E14 ventral mesencephalic tissue 
from WT (Val/Val) Sprague-Dawley rats was dissected and transplanted into homozygous 
rs6265  Met/Met  host  rats.  (c)  Following  cell  transplantation,  subcutaneous  osmotic 
minipumps  containing either mature  BDNF or PBS  were  implanted  under  the  skin  and 
attached  to  cannulas  that  were  placed  above  the  grafted  cells.  (d)  Table  including 
subsequent  treatment  of  experimental  groups  and  final  group  sizes. Abbreviations:  6-
OHDA  =  6-hydroxydopamine,  amph-induced  =  amphetamine-induced,  LD  =  levodopa, 
VM = ventral mesencephalic, GID = graft-induced dyskinesia.  

255 

 
 
 
Amphetamine-Induced Rotational Behavior 

Amphetamine-mediated rotational behavior was utilized as a method to assess 

both lesion status following 6-OHDA surgeries and graft function and dysfunction (i.e., 

GID described below) following transplantation surgeries because it is a reliable 

measure of nigrostriatal DA depletion and graft-derived DA release (Collier et al., 1999, 

2015; Dunnett & Torres, 2011; Soderstrom et al., 2008). Two weeks after lesion surgery, 

amphetamine-mediated rotational behavior was first assessed to confirm lesion status. 

Amphetamine sulfate (2.5 mg/kg) was injected (i.p.) into each subject. Rotational 

behavior was then monitored for a total of 90 minutes using the automated Rotameter 

System (TSE-Systems, Chesterfield, MO, USA). Rats that rotated 5 ipsilateral turns per 

minute or more over the 90 minute time course were included for the continuation of the 

study. Amphetamine rotations were also quantified manually at one-minute intervals in 

the rat’s home cage at five and 10 weeks post-engraftment as a secondary 

measurement of graft function. 

Levodopa Administration and LID ratings 

Four weeks after lesion surgeries, rats were primed with daily (M-F) levodopa (12 

mg/kg, 1:1 levodopa/benserazide, subcutaneous (s.c.) administration) for a total of four 

weeks before grafting surgeries. Levodopa was withdrawn one week following 

transplantation surgeries to prevent any potential toxic interactions between the grafted 

cells and levodopa (Collier et al., 2015; Steece-Collier et al., 1990). Levodopa was 

introduced again after the one-week hiatus and continued daily throughout the 

remainder of the experiment.  

256 

 
The well-established rat model of LID was employed as an indicator of graft 

function as this behavioral side effect can be ameliorated by DA neurons grafts in 

parkinsonian rats (Lane et al., 2006; Lee et al., 2000; Maries et al., 2006; Mercado et 

al., 2021; Soderstrom et al., 2008, 2010) and individuals with PD (Hagell & Cenci, 

2005). LID were evaluated on pre-graft days 1, 7, 14, and 21, and at five post-graft 

timepoints including week 2, 4, 6, 8, and 10. The rating scale employed for LID severity 

was developed by our laboratory based on specific criteria comparable to attributes of 

dyskinesia (see (Caulfield et al., 2021; Maries et al., 2006) for details). A blinded 

investigator evaluated LID behavior at one-minute intervals 20, 70, 120, 170, and 220 

minutes following levodopa injection as previously detailed (Mercado et al., 2021). A 

total LID severity score was calculated for each animal at each rating session as 

previously detailed in (Mercado et al., 2021).  

Preparation of Donor Tissue and Cell Transplantation 

Following levodopa priming, rats were assigned to either DA-grafted or non-

grafted BDNF- or PBS-infused groups based on their mean pre-grafted LID severity 

scores, ensuring that pre-graft LID mean scores were statistically similar between all 

four treatment groups. Rats in the DA-grafted groups received an intrastriatal 

transplantation of 200,000 VM cells from embryonic day 14 (E14) timed-pregnant WT 

donors. Prior to surgery, the VM tissue was collected in cold calcium-magnesium free 

(CMF) buffer; cells were then dissociated as previously detailed in (Collier et al., 2015; 

Mercado et al., 2021). Briefly, the dissected tissue was incubated for 10  minutes at 

37°C in CMF buffer containing 0.125% trypsin. Cells were then triturated with 0.005% 

DNase using a 2.0 mm tip Pasteur pipette and further triturated with a sterile 3cc, 22-

257 

 
gauge syringe. The resulting cell suspension was layered onto sterile fetal bovine serum 

(FBS) and centrifuged at 1,000 rpm for 10 minutes at 4°C, then resuspended in 1.0 mL 

Neurobasal medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cell number 

and viability were evaluated with the trypan blue exclusion test, and the final cell 

suspension concentration was adjusted to 33,333 cells/L. Cells were kept on ice during 

surgery and transplanted within five hours of preparation. The cells were injected at a 

single rostral-caudal striatal site (0.2 mm anterior, 3.0 mm lateral to bregma), with 

injections at three dorsal-ventral coordinates corresponding to 5.7, 5.0, and 4.3 mm 

ventral to the skull base (Collier et al., 2015; Mercado et al., 2021). At each coordinate, 

2 µL (injected at 0.5 µL/min) of the VM cell suspension was delivered, for a total of 6 µL 

per rat. Rats in the non-grafted group did not receive any cell suspension based on the 

logistics of transplanting cells within the 5-hour post-preparation period restraint, along 

with the necessity of implanting cannulas and minipumps. 

Intrastriatal BDNF Infusions 

In the same grafting surgical session described above, an infusion cannula was 

stereotaxically inserted to 0.3 mm dorsal of the transplanted cells (per Yurek et al., 

1996/98). The cannula (Alzet® Brain Infusion Kit 2) was attached with tubing to a 

primed 28-day Alzet® minipump (model 2004; flow rate of 0.25 µL/hour) that was then 

implanted into the subdermal intrascapular space. Prior to implantation, minipumps 

were primed for 48 hours in sterile 0.9% saline before being filled with either sterile PBS 

or 1.25 µg/µL of recombinant human BDNF (R&D Systems, Inc. Bio-Techne Corporation 

catalog # 11166-BD) dissolved in sterile PBS, similar to what has been previously 

described in (Yurek, 1998; Yurek et al., 1996). Cannulas were then permanently fixed to 

258 

 
the skull using dental cement and anchor screws that were placed into the skull earlier 

in the surgery. Minipumps were surgically removed in a sterile environment following 

completion of BDNF or PBS infusion for a total infusion exposure of four weeks. 

Effective delivery of recombinant BDNF from osmotic minipumps into a rat model has 

been demonstrated successfully (Yurek, 1998; Yurek et al., 1996) and (Altar et al., 

1994).  

Graft-induced Dyskinesia (GID) Ratings 

Amphetamine was utilized to assess graft-induced dyskinesia (GID); rats 

received a single 2 mg/kg i.p. dose of amphetamine sulfate. This method of 

amphetamine-mediated GID behavior is based on evidence that DA-grafted, but not 

sham-grafted, animals demonstrate dyskinetic behavior in response to low-dose 

amphetamine administration (Lane et al., 2009; Shin et al., 2012; Smith et al., 2012). 

This behavior, which appears phenotypically similar to LID, was rated by a blinded 

investigator using the same method and rating scale as was described for LID. GID 

were evaluated one week following minipump removal (i.e., week five post-engraftment) 

to prevent any acute effects of BDNF infusion on GID behavior. At 10 weeks post-

engraftment, GID were evaluated again; this time 24 hours prior to sacrifice. 

Necropsy 

Rats were sacrificed as detailed in Mercado et al., 2021. Briefly, 

phenytoin/pentobarbital euthanasia solution (250 mg/kg; i.p., VetOne, Boise, ID, USA) 

was used to deeply anesthetize the rats. Rats then underwent intracardiac perfusions of 

room temperature heparinized 0.9% saline followed by cold 4% paraformaldehyde. After 

perfusion was completed, brains were removed and placed in 4% paraformaldehyde for 

259 

 
a total of 24 hours at 4°C. Brains were then submersed in 30% sucrose at 4°C until 

sectioning. For sectioning, brains were cut coronoally using a sliding microtome at a 

thickness of 40 µm. Brain sections were stored in cryoprotectant solution at -20°C.  

Histology 

TH graft Cell Number and Volume 

Briefly, tissue sections were rinsed in Tris-buffered saline containing 0.3% Triton-

X (TBS-Tx). Sections were incubated in 0.3% hydrogen peroxide, then blocked in 10% 

normal goat serum (NGS) for 90 minutes. For primary antibody incubation, tissue 

sections were incubated overnight at room temperature with rabbit anti-TH (see Table 

4.1). Following primary incubation, sections were incubated in biotinylated goat anti-

rabbit secondary antibody (Table 4.1), then developed using avidin/biotin enzyme 

complex.  

A blinded investigator used the Stereo Investigator® Optical Fractionator method 

(MBF Bioscience, Williston, VT, USA) to quantify TH-positive (TH+) cells within the 

grafted striatum. The 20x objective (numerical aperture 0.75) was used to count cells on 

a Nikon Eclipse 80i microscope with a 200 µm x 200 µm counting frame. The optical 

dissector height was set to 20 µm, and the guard zone was set to 2.0 µm. This method 

was completed in 4-12 serial (1:6) TH+ section in which the number of sections varied 

depending on the rostral-caudal extent of the graft.  

Using the same tissue sections for total enumeration, a blinded investigator 

employed the Stereo Investigator® Cavalieri Estimator to quantify graft volume. 

Contours were traced around the central region of the graft, and then a grid with random 

260 

 
sampling sites (50-µm spacing) was superimposed over the contours. Collected data 

were expressed as total estimated graft volume (mm3). 

Neurite Outgrowth 

Two-dimensional (2D) images fluorescently labeled for TH, at 4x magnification, 

were saved as .tiff files and imported into Fiji image processing package. Eight total 

regions of interest (ROIs) were created measuring 600 µm2. Four ROIs were first placed 

around the edge of the perimeter of the grafted TH+ cells. This was considered the 

proximal region, including proximal dorsolateral, dorsomedial, ventrolateral, and 

ventromedial. The ROIs were placed in this way around the graft in order to avoid the 

cannula injection site located immediately dorsal from the graft. An additional four ROIs 

were placed 625 µm from the edge of the grafted TH+ cell bodies. This was considered 

the distal regions, including distal dorsolateral, dorsomedial, ventrolateral, and 

ventromedial. ROIs were then added into the ROI manager, converted to 8-bit, and 

inverted from the original fluorescent color. The background of each image was 

removed and the contrast was enhanced for optimal analysis. Each image is then made 

into a binary, and the threshold function is applied. All white areas that were TH+ were 

measured for threshold amount and recorded. Data are reported as average neurite 

density in pixels2 for proximal and distal regions, and distal neurite density in pixels2 for 

each region surrounding the graft (i.e., dorsolateral, dorsomedial, ventrolateral, and 

ventromedial). These orientations were used to avoid the area of the striatum that was 

compromised by the overlying cannula (Figure 4.2f). This procedure was adapted from 

(Quintino et al., 2022).  

261 

 
Immunofluorescence 

DAB-chromogenic TH-labeled sections as described above were used as a guide 

when choosing one representative grafted striatal section for each immunofluorescent 

assay. For all immunohistochemical procedures, tissue sections were rinsed in TBS-Tx, 

blocked in 10% NGS/0.3% TBS-Tx, and then incubated overnight at 4°C. Tissue 

sections were then labeled with their respective Alexa Fluor™ secondary antibodies 

(1:500 dilution; see Table 4.1) for 90 minutes at room temperature and protected from 

light. Sections were mounted and coverslipped with Vectashield® anti-fade mounting 

medium with DAPI (H-1500; Vector Laboratories, Inc. Burlingame, CA, USA). 

Fluorescent Image Acquisition 

Using a Nikon A1 laser scanning confocal system equipped with a Nikon Eclipse 

Ti microscope and Nikon NIS-Elements AR software, all confocal images of (1024 x 

1024) immunofluorescent stained tissue sections were acquired. For DAT/TH IHC 

experiments, the 4x objective was used to collect a full image of the entire graft from 

each tissue section. One image of the contralateral intact striatum was also taken for 

comparison. Z-stacks of 2 µm and a scan speed of 1/8 frame/second were used. For 

VGLUT2/VMAT2 colocalization experiments, z-stacks were acquired through the entire 

thickness of the mounted tissue sections using the 60x oil-immersion objective 

(numerical aperture 1.40). A z-step of 1.5 um was used with a scan speed of 1/8 

frame/sec. Two images of each section were taken of the dorsolateral area of the graft 

because the dorsolateral striatum is a major input region of the basal ganglia and 

functions predominantly in motor control. Again, one image of the intact side was also 

taken for comparison. Lastly, for Iba1/GFAP/TH IHC experiments, z-stacks were 

262 

 
acquired using the 10x objective in which multiple images were taken in order to capture 

the entire graft region in the striatum. Z-steps were 2 um and the scan speed was 1/8 

frame/sec. Additional 10x images of the intact striatum was also taken for comparison. 

Table 4.1: Targeted Antigens and corresponding antibodies. 
Secondary antibody catalog numbers are Alexa Fluor®-conjugated, purchased from 
Invitrogen®. 

263 

 
 
 
 
Imaris Fluorescent Image Quantification 

Dual-label protein analysis of VGLUT2 and TH 

3-dimensional (3D) z-stacks of grafted tissue fluorescently labeled for VGLUT2 

and TH proteins were imported into Imaris®, converted to the native Imaris® file format, 

and subtracted of any background fluorescence. The surface function was used to 

generate a 3D reconstruction of the TH+ neuron fibers in the graft (µm3). The spots 

function then was used for VGLUT2 protein puncta, taking care to maintain the same 

parameters across all images. VGLUT2 puncta inside the TH+ grafted surface were 

filtered through Object-Object statistics using the “Shortest Distance to Surface” function 

to select only those located within the TH surface. Data are presented as the number of 

VGLUT2 protein puncta inside the grafted TH surface (µm³). 

Dual-label immunohistochemical DAT and TH protein analysis 

Two-dimensional (2D) confocal images of tissue immunolabeled for TH and the 

dopamine transporter (DA) proteins were imported into Imaris® and converted into the 

native Imaris® file format. Background subtraction of each image was conducted in 

order to minimize any background fluorescence in each fluorescent channel. The 

surface function was used to create an accurate reconstruction of both TH and DAT 

fibers within the graft. Data are represented as the ratio of DAT fluorescent intensity 

sum/DAT surface area (µm2) to TH fluorescent intensity sum/TH surface area (µm2). 

Triple-label immunohistochemical Iba1/GFAP/TH protein analysis 

3D z-stacks of grafted brain sections immunolabeled for TH, Iba1, and GFAP 

proteins were imported into Imaris® and converted to the native Imaris®  file format. 

Background subtraction of each image was conducted to minimize any background 

264 

 
fluorescence. 3D surface objects of TH, Iba1, and GFAP were created using semi-

automatic thresholding and the surface function plugin. Data are reported as Iba1 

surface volume (µm3) normalized to the surface volume of the graft (TH; µm3). GFAP is 

reported in a comparable manner. 

Triple-label immunohistochemical VMAT2/VGLUT2/TH protein analysis 

3D z-stacks of grafted tissue labeled for VMAT2, VGLUT2, and TH proteins were 

imported into Imaris® and converted to the native Imaris® file format. Background 

subtraction of each image was conducted to minimize any background fluorescence. As 

before, the surface function was used to generate an accurate 3D reconstruction of the 

TH+ neuron fibers in the graft (µm3). The spots function was then used for both VMAT2 

and VGLUT2 protein puncta, maintaining the same parameters across all images. The 

MATLAB “Colocalization” plugin was used to find VMAT2 and VGLUT2 protein puncta 

that were “co-localized” within 0.5 µm of each other. The co-localized VMAT2/VGLUT2 

puncta were then filtered using the Object-Object statistics “Shortest distance to 

Surface” to include only co-localized puncta that were within the TH surface. Data are 

represented as the number of co-localized VMAT2/VGLUT2 protein puncta inside the 

grafted TH surface (µm3). 

ELISA Assay for Interleukin-6 (IL-6) 

The Rat IL-6 ELISA kit from Invitrogen (catalog number BMS625) was used for 

IL-6 analysis. During perfusions at the conclusion of the study (10 weeks post-

engraftment), cardiac punctures from the right atrium of the heart were performed to 

collect blood samples from each rat subject. Blood samples were subsequently spun 

down using a centrifuge at 2200 rpm for 10 minutes at 4°C. The plasma serum was 

265 

 
collected and stored at -80°C until processing. Plasma samples were prepared and 

diluted prior to ELISA according to the manufacturer’s instructions.  

Statistical Analysis  

All behavioral data (i.e., GID and LID) were analyzed using non-parametric 

statistics including the Kruskal-Wallis test with Dunn’s multiple comparisons or Mann-

Whitney U tests with Dunn’s multiple comparisons (between subject comparisons) as 

LID and GID behavioral data are created using an ordinal rating scale. This statistical 

test was also employed for the results collected from the IL-6 ELISA as the standard 

deviation (SD) was significantly different between groups.  Amphetamine-mediated 

rotations were analyzed using a one-way ANOVA with Tukey’s multiple comparisons 

test.  

Unpaired two-tailed t-tests were used to compare average neurite outgrowth 

(proximal and distal) surrounding the DA grafts between treatment groups (BDNF- vs. 

PBS-infused animals). A two-way ANOVA with Tukey’s multiple comparisons was used 

to analyze distal neurite outgrowth in each region from the graft (i.e., DL, DM, VL, VM). 

Unpaired two-tailed t-tests were employed for DAT:TH intensity sum/µm3 expression 

and VMAT2/VGLUT2 colocalization in TH+ neurons. This statistical test was also used 

to compare Iba1 volume (µm3)/number of TH+ neurons and GFAP volume 

(µm3)/number of TH+ neurons. A one-way ANOVA with Sidak’s multiple comparisons 

was used for Iba1 or GFAP surface volume (µm3) alone.  

Non-parametric Spearman correlation tests were applied for all correlations with 

GID behavior. Amphetamine rotation correlations were analyzed using Pearson 

correlation. Statistical outliers, while rare, were identified using ROUT and Grubb’s 

266 

 
outlier tests. Parametric statistical tests were chosen for analysis only when data met 

assumptions for normality and homogeneity of variances. All statistical analysis were 

completed using GraphPad Prism software for Windows (v. 10.4.1).  

267 

 
 
 
RESULTS 

Exogenous BDNF infusion into DA-grafted animals enhances functional graft 

efficacy (i.e., amelioration of LID) and neurite outgrowth 

We hypothesized that exogenous BDNF infusion into DA-grafted homozygous 

rs6265 (i.e., Met/Met) animals would generate enhanced behavioral recovery from LID 

earlier than the other grafted and non-grafted groups. As expected, in comparison to the 

non-grafted BDNF-infused subjects, BDNF administration to DA graft recipients led to 

faster amelioration of LID behavior compared to the DA-grafted PBS-infused 

parkinsonian rats. Specifically, grafted BDNF-infused rats demonstrated a significant 

reduction in LID behavior compared to non-grafted BDNF-infused animals by four 

weeks post-engraftment. In contrast, grafted PBS-infused animals did not exhibit a 

significant amelioration of LID severity until considerably later at week 10 post-

engraftment (Figure 4.2a. Week 4: p = 0.0421 gBDNF vs. non-grafted BDNF; Week 10: 

p = 0.0032 gBDNF vs. non-grafted BDNF, p = 0.0176 gPBS vs. non-grafted PBS). At the 

final week (week 10), grafted BDNF- and PBS-infused animals exhibited approximately 

the same LID severity scores (Figure 4.2a; p ≥ 0.9999 gBDNF vs. gPBS at week 10).  

Because BDNF is a protein critical for neuronal survival, maturation, and function 

(Gonzalez et al., 2016; Hyman et al., 1991; Kowiański et al., 2018; Lai & Ip, 2013; Park 

& Poo, 2013; Sasi et al., 2017; Zagrebelsky et al., 2020), we investigated whether 

BDNF infusion impacted the size of the graft or the number of surviving grafted neurons. 

While no statistical significance was apparent (Figure 4.2d; p = 0.1449 gBDNF vs. 

gPBS), there was an inclination toward an increase in number of surviving DA neurons 

in the grafted BDNF-infused animals (Figure 4.2d). Likewise, a similar, but even 

268 

 
slighter, trend was demonstrated in graft size/volume with the grafted BDNF-infused 

subjects having a slightly larger graft volume, albeit not statistically significant (Figure 

4.2e, p = 0.3347 gBDNF vs. gPBS, not significant). In addition to graft volume and 

number of grafted neurons, I also analyzed average TH+ neurite area as an indication 

of graft-derived outgrowth. Again, we had hypothesized that BDNF administration would 

enhance neurite outgrowth in the DA-grafted parkinsonian rats due to the known 

function of BDNF and based on previous findings (see (Yurek, 1998; Yurek et al., 

1996)). Data revealed a significant increase in average neurite outgrowth in the DA-

grafted BDNF-infused compared to the DA-grafted PBS-infused animals, both proximal 

and distal to the graft (Figure 4.2g, Proximal: p = 0.0367 gBDNF vs. gPBS; Distal: p = 

0.0175 gBDNF vs. gPBS). Additionally, when reported as distal neurite density alone, 

the dorsolateral region of the graft in the DA-grafted BDNF-infused animals 

demonstrated significantly increased neurite density compared to the other regions 

(Figure 4.2h, DL vs. DM: p = 0.0391, DL vs. VL: p = 0.0116, DL vs. VM: p = 0.0034 in 

gBDNF) and compared to DA-grafted PBS-infused animals (p = 0.0006 DL gBDNF vs. 

DL gPBS). 

269 

 
Figure 4.2: Impact of BDNF supplementation on LID behavior and neurite 
outgrowth. 
(a) Total LID severity scores for grafted and non-grafted treatment groups throughout the 
pre-graft  period  and  for  10  weeks  post-graft  behavioral  recovery.  Statistics:  Non-
parametric Kruskal-Wallis test with Dunn’s multiple comparisons test at each time point.  

270 

 
 
 
 
 
Figure 4.2 (cont’d) 
Week 4: *p = 0.0421 gBDNF vs. non-grafted BDNF. Week 8: *p = 0.0169 gBDNF vs. non-
grafted BDNF. Week 10: **p = 0.0032 gBDNF vs. non-grafted BDNF; *p = 0.0176  gPBS 
vs. non-grafted PBS. Non-grafted groups (BDNF vs. PBS) were not significantly different 
at any post-graft time points (p ≥ 0.9324 for all time points). Grafted groups (BDNF- and 
PBS-infused) were not significantly different at any post-graft time points (p ≥ 0.9319 for 
all time points). (b) LID severity over the 220-minute time course for each animal response 
at week 4, 6, 8, and 10 post-engraftment. Statistics: Non-parametric Kruskal-Wallis test 
with Dunn’s multiple comparisons tests at each time point.  Week 4: (20 minutes): *p = 
0.0129 grafted BDNF vs. non-grafted BDNF; (70 minutes) *p = 0.0130 gBDNF vs. non-
grafted BDNF;  (120  minutes) *p  =  0.0184 gBDNF vs. non-grafted  BDNF; Week 6:  (70 
minutes) *p = 0.0314 gBDNF vs. non-grafted BDNF, *p = 0.0189 gPBS vs. non-grafted 
PBS; (120 minutes) **p = 0.0047 gBDNF vs. non-grafted BDNF, *p = 0.0307 gPBS vs. 
non-grafted PBS; (170 minutes) *p = 0.0368 gBDNF vs. non-grafted BDNF; Week 8: (70 
minutes) **p = 0.0016 gBDNF vs. non-grafted BDNF; (120 minutes) **p = 0.0015 gBDNF 
vs. non-grafted BDNF, *p = 0.0259 gPBS vs. non-grafted PBS; Week 10: (70 minutes) 
**p = 0.0018 gBDNF vs. non-grafted BDNF, *p = 0.0390 gPBS vs. PBS; (120 minutes) 
**p = gBDNF vs. non-grafted BDNF, *p = 0.0376 gPBS vs. non-grafted PBS (c) Total LID 
score for each treatment group showing each individual animal response at weeks 4, 6, 
8,  and  10.  Statistics:  Non-parametric  Kruskal-Wallis 
test  with  Dunn’s  multiple 
comparisons test at each time point. (d) Stereologically estimated total number of grafted 
DA  neurons.  Statistics:  Mean  ±  SEM.  Unpaired  two-tailed  t-test,  not  significant.  (e) 
Stereologically  estimated  total  grafted  volume.  Statistics:  Mean  ±  SEM.  Unpaired  two-
tailed t-test, not significant. (f) Representative confocal fluorescent micrograph of the DA-
grafted striatum in the Met/Met host parkinsonian rats. Magnification at 4x, scale bar = 
300 µm. The cannula placement is depicted in the dorsal region of the striatum above the 
grafted  DA  neurons.  Each  numbered  box  represents  the  analysis  region  for  neurite 
outgrowth  of TH+  DA  fibers.  1  =  Proximal dorsomedial,  2  =  Proximal  dorsolateral,  3  = 
Proximal  ventrolateral,  4  =  Proximal  ventromedial,  5  =  Distal  dorsomedial,  6  =  Distal 
dorsolateral, 7 = Distal ventrolateral, 8 = Distal ventromedial.  

271 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4.2 (cont’d) 
(g)  Average  neurite  density  of  total  fibers  surrounding  cell  bodies  of  DA  graft,  both 
2
.  Statistics:  Mean  ±  SEM. 
proximal  and  distal  to  the  graft.  Data  are  reported  as  pixels
2
) 
Unpaired two-tailed t-test between proximal and distal. (h) Distal neurite density (pixels
of each region (DL, DM, VL, VM) surrounding the graft. Statistics: Mean ± SEM. Two-way 
ANOVA  with  Tukey’s  multiple  comparisons.  Abbreviations:    LID  =  levodopa-induced 
dyskinesia, BDNF = brain-derived neurotrophic factor, LD = levodopa, DA = dopamine, 
DL = dorsolateral, DM = dorsomedial, VL = ventrolateral, VM = ventromedial.  

272 

 
 
 
 
 
Exogenous BDNF administration increased the severity and incidence of GID in 

DA-grafted homozygous rs6265 (Met/Met) rats 

In contrast to our hypothesis, DA-grafted BDNF-infused animals exhibited 

significantly higher GID severity when compared to DA-grafted PBS-infused animals at 

week 5 post-engraftment (Figure 4.3a, p = 0.0193 gBDNF vs. gPBS). While this 

significant difference was lost at 10 weeks post-engraftment, a similar trend was 

retained with a slightly higher GID severity in the DA-grafted BDNF-infused rats (Figure 

4.3a, p = 0.0991 gBDNF vs. gPBS). To complement the GID severity results, I also 

examined the incidence of GID behavior in both grafted groups. Total GID incidence 

was defined as the number of animals in each group that demonstrated a total GID 

rating score of 4 or higher. I have additionally included the incidence of peak 

amphetamine-mediated GID behavior which we defined as the number of animals in 

each group with a peak (70 minute timepoint) GID score as 2 or higher. The incidence 

scores of GID (total and peak) were determined accordingly because a total score of <4 

and a peak score <2 are reflective of stereotypic behaviors that can occur in non-

grafted/non-lesioned rats (e.g., intermittent licking and chewing).  

At 5 weeks post-engraftment, DA-grafted BDNF-infused animals had a much 

greater percent incidence of both total and peak GID behavior in comparison to the DA-

grafted PBS-infused animals (Figure 4.3b; 55.6% compared to 11.1% in total GID, and 

66.7% to 22.2% in peak GID incidence). Likewise, at 10 weeks post-engraftment, 

percent incidence of GID behavior in the grafted BDNF-infused animals was 44.4% total 

GID compared to 22.2% total in grafted PBS-infused animals, and 33.3% peak GID 

compared to 22.2% peak in grafted PBS-infused animals (Figure 4.3c).  

273 

 
Mercado and colleagues demonstrated a statistically positive correlation between 

total GID severity and the expression of VGLUT2 within grafted DA neurons only in the 

homozygous rs6265 (Met/Met) animals engrafted with WT eVM cells (Mercado et al., 

2021). VGLUT2 inside DA neurons is atypical and indicative that the grafted DA neurons 

are co-releasing glutamate (El Mestikawy et al., 2011). In embryonic stages, DA 

neurons co-express VGLUT2; however, as the neurons mature, their immature 

phenotype of VLGUT2 co-expression is lost (El Mestikawy et al., 2011; Kordower et al., 

1996) for the most part (Kawano et al., 2006; Morales & Root, 2014; Yamaguchi et al., 

2015). In our study, we hypothesized that administering exogenous BDNF would induce 

the maturation of the WT DA neurons grafted into homozygous Met/Met rats, therefore 

decreasing the expression of VGLUT2 and ameliorating GID behavior correlated with 

this marker (Mercado et al., 2021). However, our results showed that VGLUT2 

expression is maintained even after exogenous administration of BDNF in the DA-

grafted animals. Indeed, no statistical differences were found between grafted BDNF- 

and grafted PBS-infused animals (Figure 4.3e, p = 0.7422 gBDNF vs. gPBS). 

Unexpectedly, VGLUT2 expression was also no longer correlated with GID behavior 

(Figure 4.3f, r = 0.6303, p = 0.0751 gBDNF; r= -0.07207, p = 0.8889 gPBS), although 

there was a positive trend in the DA-grafted BDNF-infused group.  

274 

 
Figure 4.3: Impact of BDNF supplementation of GID behavior. 
(a)  Total  amphetamine-induced  GID  severity  scores  at  week  5  and  week  10  post-
engraftment. Statistics: Mean ± SEM. Unpaired two-tailed t-tests between gBDNF and  

275 

 
 
Figure 4.3 (cont’d) 
gPBS groups. Week 5: p = 0.0193 gBDNF vs. gPBS. Week 10: p = 0.0991. (b) Percent 
incidence of total GID severity score of ≥ 4 in all four treatment groups at week 5 and 
week  10  post-engraftment.  Data  expressed  as  Mean  ±  SEM.  (c)  Percent  incidence  of 
peak (70 minutes post-amphetamine administration) GID severity score of ≥ 2 in all four 
treatment  groups  at  week  5  and  week  10  post-engraftment.  (d)  Schematic  diagram 
depicting  synaptic  connectivity  and  VGLUT2  expression  in  immature  (embryonic)  and 
mature  dopaminergic  neurons.  Immature  DA  neurons  express  VGLUT2  and  form 
asymmetric,  atypical  connections  directly  onto  the  dendritic  head  of  MSNs.  As  the 
neurons  mature,  they  lose  the  VGLUT2  phenotype  and  form  typical  en  passant 
associations onto the shaft of the dendritic spine of MSNs (El Mestikawy et al., 2011). (e) 
Fluorescent  micrograph  and  subsequent  Imaris  3D  reconstruction  of  DA  (THir)  fibers 
containing VGLUT2 protein. Scale bar = 5 um. (f) Quantification of the number of VGLUT2 
3
protein found within TH+ DA fibers, normalized to the TH surface volume (um
). Statistics: 
Mean ± SEM. Unpaired two-tailed t-tests between gBDNF and gPBS treatment groups. 
(g)  Spearman  correlation  between  quantify  of  VGLUT2  protein  located  inside  TH+ 
neurons  and  total  amphetamine-mediated  GID  score  at  week  5  post-engraftment.  No 
significance. Data for week 10 post-engraftment was also not significant: data not shown. 
Abbreviations:  GID  =  graft-induced  dyskinesia,  VGLUT2  =  vesicular  glutamate 
transporter  2,  DA  =  dopamine,  MSNs  =  medium  spiny  neurons,  TH  =  tyrosine 
hydroxylase. 

GID behavior is associated with behavioral and morphological indices of excess 

DA release in DA-grafted BDNF-infused animals  

In a 6-OHDA-lesioned parkinsonian rat, the subject normally rotates ipsilateral, or 

in the same direction, toward the lesioned hemisphere upon administration of 

amphetamine which causes DA release from intact DA terminals in the intact 

contralateral striatum (Figure 4.4a, see (Dunnett & Torres, 2011)). Amphetamine, an 

indirect DA agonist, will bind to monoamine transporters, thereby increasing the release 

of DA into the synapse from intact nigrostriatal DA terminals (or grafted DA neurons). 

Since one hemisphere is lesioned in our unilaterally lesioned rat model, amphetamine 

will only activate increased DA release from the intact hemisphere, causing the animal 

to rotate ipsilaterally (Dunnett & Torres, 2011). The DA graft should mitigate rotation 

behavior if equal release of DA occurs between the two striatal hemispheres. However, 

276 

 
 
if the graft is releasing more DA than the intact striatum, the rat will rotate contralaterally, 

or away from, the lesioned side after amphetamine administration.  

As expected, there were no differences in net ipsilateral amphetamine rotations 

per minute (Figure 4.4b; p = 0.4297 Week 5, p = 0.9842 Week 10 non-grafted BDNF 

vs. non-grafted PBS) or in total ipsilateral rotations over 220 minutes post-amphetamine 

(Figure 4.4cd; p = 0.9764 Week 5, p > 0.9999 Week 10 non-grafted BDNF vs. non-

grafted PBS) found between the sham-grafted (BDNF- and PBS-infused) animals at 

either week 5 or week 10 of the study. Both grafted BDNF-infused and grafted PBS-

infused parkinsonian rats demonstrated recovery of amphetamine-mediated rotational 

behavioral following engraftment at week 5 (Figure 4.4b, p <0.0001 gBDNF vs. non-

grafted BDNF; p = 0.0071 gPBS vs. non-grafted PBS) and week 10 (p <0.0001 gBDNF 

vs. non-grafted BDNF, p <0.0001 gPBS vs. non-grafted PBS). When comparing DA-

grafted BDNF- and PBS-infused animals, there was a significant increase in the number 

of ipsilateral rotations in the PBS-infused animals (i.e., increased contralateral rotations 

in the BDNF-infused animals) at week 5 (Figure 4.4b per minute: p = 0.0159; Figure 

4.4c total: p = 0.0307 gBDNF vs. gPBS). Although significance is lost at week 10 

between these groups, there remains a similar trend between the grafted BDNF- and 

grafted PBS-infused animals in which the BDNF-infused animals have a greater number 

of contralateral rotations, suggesting that excess DA is being released from the DA-

grafted rats there were exposed to exogenous BDNF infusion (Figure 4.4d).  

In order to further assess whether the DA-grafted BDNF-infused animals have a 

propensity for increased DA release, immunohistochemical postmortem expression of 

DAT was examined. DAT is a transmembrane protein responsible for clearing DA from 

277 

 
the extracellular space; an increase in DAT has been linked to an increase in DA 

release since DAT upregulation is required in order to clear higher concentrations of DA 

from the synapse (Lohr et al., 2017; Zhu & Reith, 2008). Affirmatively, there was a 

significant increase in DAT expression per TH+ neuron (i.e., DAT:TH Intensity/um2) in 

the grafted BDNF-infused animals (Figure 4.4f, p = 0.0174 gBDNF vs. gPBS). 

Fluorescent intensity of DAT immunohistochemical staining here is synonymous with 

expression of the DAT protein as the staining pattern of DAT fills the entire TH+ neuron. 

Strikingly, expression levels of DAT:TH were positively and robustly, correlated with GID 

behavior at week 5 post-engraftment (Figure 4.4g; r = 0.8320, p = 0.00716 gBDNF). 

This correlation was no longer significant at week 10 (data not shown), although GID 

behavior was also not significant at this timepoint.  

DAT:TH expression and ipsilateral rotations were investigated to determine any 

correlation of these measures. While not statistically significant, DAT:TH expression 

seemed to have a negative trend with net ipsilateral rotations at week 5 post-

engraftment: the animal with the highest DAT:TH expression had the lowest number of 

ipsilateral rotations (or highest contralateral rotations) only in the grafted BDNF-infused 

group (Figure 4.4hi). When DAT and TH expression were examined separately and 

then correlated to net ipsilateral rotations, a similar negative trend was apparent in the 

grafted BDNF-infused animals in which a higher DAT expression denoted a lower 

number of ipsilateral rotations (Figure 4.4hii). In contrast, with TH+ expression alone, 

no significant correlation or trend existed between TH and the number of rotations 

(Figure 4.4hiii), suggesting that DAT expression alone is more likely associated with 

278 

 
the number of rotations a unilaterally lesioned parkinsonian rat makes in response to 

amphetamine administration.  

Recent evidence has established that VMAT2 is co-expressed with VGLUT2 in a 

subpopulation of midbrain DA neurons in the ventral tegmental area (VTA) and the 

SNpc (Hnasko et al., 2010; H. Shen et al., 2021). Therefore, this indicates that a 

subpopulation of DA neurons can co-release DA and glutamate from terminals in the 

striatum (Hnasko et al., 2010; H. Shen et al., 2018). Furthermore, this lends to the 

theory of vesicular synergy, as introduced above, which posits that, if VMAT2 and 

VGLUT2 are co-localized on the same synaptic vesicle, the presence of VGLUT2 

(glutamate) will increase the pH gradient by acidifying the inside of the synaptic vesicle, 

allowing for more loading of DA (Buck et al., 2021; Hnasko et al., 2010; H. Shen et al., 

2018).  

In the context of our studies based on this theory, I hypothesized that VMAT2 and 

VGLUT2 are co-localized on the same synaptic vesicles, increasing the amount of DA 

that is loaded, thereby increasing the amount of vesicular DA release and GID behavior 

in the grafted BDNF-infused animals. As such, I have examined whether the (presumed) 

co-localization of VMAT2 and VGLUT2 exists inside TH+ DA fibers. Although no 

significant difference were found between the DA-grafted BDNF-infused and DA-grafted 

PBS-infused animals, there was a slight trend of increased VMAT2/VGLUT2 presumed 

colocalization in the grafted BDNF-infused animals (Figure 4.4j, p = 0.1758). More 

importantly, however, the number of (presumed) co-localized VMAT2/VGLUT2 inside 

TH+ fibers was significantly correlated with GID behavior in the grafted BDNF-infused 

animals. Specifically, an increase of GID behavior correlated to an increase in 

279 

 
(presumed) VMAT2/VGLUT2 colocalization inside TH+ fibers (Figure 4.4k; r = 0.7647, p 

= 0.02050 gBDNF). To my knowledge, this is the first evidence suggestive that VMAT2 

and VGLUT2 are co-localized in the same vesicle in grafted eVM DA neurons.  

Figure 4.4: Exogenous BDNF administration is associated with indices of excess 
DA release. 
(a) Schematic depicting the amphetamine-mediated rotational behavior of a unilaterally 
6-OHDA-lesioned animal. Upon amphetamine administration, a lesioned animal will  

280 

 
 
Figure 4.4 (cont’d) 
rotate  ipsilateral  (same  side)  toward  the  lesioned  striatum.  Modified  from  (Dunnett  & 
Torres, 2011). (b) Net ipsilateral rotations per minute, manually counted at the 70-minute 
post-amphetamine injection timepoint. Rotations are reported for both week 5 and week 
10  post  engraftment.  Statistics:  Mean  ±  SEM.  Two-way ANOVA  with  Tukey’s  multiple 
comparisons. Week 5 post-graft: ****p = <0.0001 gBDNF vs. non-grafted BDNF, **p = 
0.0071 gPBS vs. non-grafted PBS. #p = 0.0159 gBDNF vs. gPBS. Week 10 post-graft: 
****p < 0.0001 gBDNF vs. non-grafted BDNF, ****p < 0.0001 gPBS vs. non-grafted PBS. 
(c) Amphetamine rotational behavior expressed as net ipsilateral rotations at week 5 and 
10  (d)  post-engraftment.  Ordinary  one-way ANOVA  with  Tukey’s  multiple  comparisons 
tests. Week 5: p = 0.0307 gBDNF vs. gPBS. p = 0.0022 gPBS vs. non-grafted PBS. p = 
<0.0001  gBDNF  vs.  non-grafted  BDNF.  Week  10:  p  =  0.0514  gBDNF  vs.  gPBS.  p  = 
<0.0001 gPBS vs. non-grafted PBS. p = <0.0001 gBDNF vs. non-grafted BDNF.  

281 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4.4 (cont’d) 
(e)  (i)  Representative  confocal  fluorescent  micrograph  demonstrating  differing  staining 
patterns  of  DAT  and  TH  expression  in  the  grafted  parkinsonian  rat  striatum.  (ii) 
Fluorescent  micrographs  depicting  an  increase  in  DAT  staining  (cyan)  in  the  grafted 
parkinsonian striatum treated with BDNF administration compared to PBS treatment (iii). 
Scale bar = 300 µm. (f) Quantification of DAT expression in the grafted DA neurons. Data 
are expressed as the ratio of the sum fluorescent intensity of DAT to the sum fluorescent 
2
).  Statistics: 
intensity  of  TH,  both  normalized  to  their  respective  surface  areas  (um
Unpaired  two-tailed  t-tests.  p  =  0.0174  gBDNF  vs.  gPBS.  (g)  Spearman  correlation 
and  total  amphetamine-mediated  GID  severity  score  at 
between  DAT:TH  intensity/um
was  significantly 
week  5  and  week  10  post-engraftment.  Only  DAT:TH  intensity/um
correlated in the grafted BDNF-infused animals at week 5. p = 0.007716. Correlation for 
week 10 not shown, not significant (p = 0.1967). 

2 

2 

282 

 
 
 
Figure 4.4 (cont’d) 
(hi)  Spearman  correlation  between  DAT:TH 
amphetamine-mediated rotations. (hii) Spearman correlation between DAT intensity/um
alone.  (i)  Confocal  fluorescent  micrograph  depicting 
alone,  and  (hiii)  TH  intensity/um
(presumed) co-localization of VMAT2 and VGLUT2 protein located inside TH+ DA neuron 
fibers. Scale bar = 2 µm  

ipsilateral 
2 

intensity/um

and  net 

2 

2 

283 

 
 
 
 
 
 
 
 
 
 
Figure 4.4 (cont’d) 
(j) Quantity of number of (presumed) co-localized VMAT2/VGLUT2 protein located inside 
TH  DA  neuron  fibers.  Statistics:  Mean  ±  SEM.  Unpaired  two-tailed  t-tests.  p  =  0.1758 
gBDNF  vs.  gPBS.  (k)  Spearman  correlation  between  the  quantity  of  (presumed)  co-
localized  VMAT2/VGLUT2  protein  located  inside  TH  DA  neuron  fibers  and  total  GID 
severity  scores  at  week  5  post-engraftment.  p  =  0.02050  in  the  grafted  BDNF-infused 
animals.  

284 

 
 
 
 
 
 
Exogenous BDNF infusion increases microglial (Iba1) expression in DA-grafted 

animals  

As introduced in Chapter 3, we demonstrated previously that engrafted 

parkinsonian rats exhibited higher percentages of asymmetric synapses following 

immune activation, and that this correlated significantly with increased GID (Soderstrom 

et al., 2008). Similarly, in clinical trials, patients developed GID behavior after withdrawal 

of immune suppression (Freed et al., 2001; Hagell et al., 2002; Olanow et al., 2003). 

Because this evidence points to a possible influential role of the immune response in 

the induction of GID behavior, we investigated two well-known immune markers, Iba1 

and GFAP.  

Iba1, which is a factor that can be involved in the creation and elimination of 

synapses (Tremblay et al., 2011), was used as an indicator of inflammation and 

quantified in the striatum of all rat subjects. To label astrocytes, GFAP was employed 

and served as an additional inflammatory marker. Injury, such as grafting, can activate 

astrocytes, leading to the release of proinflammatory chemokines and cytokines 

(Alhadidi et al., 2024; Giovannoni & Quintana, 2020). Thus, along with Iba1 expression, 

elevated GFAP is also associated with inflammation.  

BDNF-infused DA-grafted animals demonstrated a slight, though not statistically 

significant, increase in overall Iba1 expression compared to the vehicle (PBS)-infused 

DA-grafted subjects (Figure 4.5b; p = 0.3806 gBDNF vs. gPBS). Similarly, in the non-

grafted animals, Iba1 was slightly increased in the BDNF-infused animals than in the 

PBS-infused group, but this difference was not statistically significant (Figure 4.5b, p = 

0.5645 gBDNF vs. non-grafted BDNF). When normalized to the number of grafted TH+ 

285 

 
neurons, Iba1 expression was significantly increased in the DA-grafted BDNF-infused 

rats compared to the grafted PBS-infused treatment (Figure 4.5c, p = 0.0232 gBDNF 

vs. gPBS). Although statistical significance was noted, there was no significant 

correlation found between Iba1 expression and GID behavior at week 5 (Figure 4.5d; r 

= -0.06723, p = 0.8685) or week 10 (data not shown).  

GFAP expression was significantly greater in the DA-grafted, compared to the 

non-grafted, BDNF-infused animals, suggesting that grafting stimulates astrocyte 

upregulation (Figure 4.5e, p = 0.0168 gBDNF vs. non-grafted BDNF). Following 

normalization to the number of grafted TH+ neurons, no significant differences were 

observed between the grafted treatment groups (Figure 4.5f, p = 0.3510 gBDNF vs. 

gPBS). Furthermore, similar to Iba1 expression, GFAP expression did not significantly 

correlate with GID behavior at either week 5 (Figure 4.5g; r = -0.03361, p = 0.9397 

gBDNF) or 10 post-engraftment (data not shown). 

To complement the immunohistochemical findings, an interleukin-6 (IL-6) 

sandwich enzyme-linked immunosorbent assay (ELISA) was conducted on serum 

collected from cardiac punctures at the conclusion of the study (week 10). IL-6 is a 

proinflammatory cytokine often elevated in response to inflammation or injury (Tanaka et 

al., 2014). The results indicated no significant differences in IL-6 concentrations among 

the non-grafted or grafted treatment groups (Figure 4.5h, p > 0.9999 for all groups). 

However, a subtle increase in IL-6 was observed in the grafted BDNF-infused rats, 

consistent with the trends seen in Iba1 and GFAP expression (Figure 4.5h). Despite 

this, no significant correlation was found between IL-6 levels and GID behavior at weeks 

286 

 
5 (Figure 4.5i; r = -1907, p = 0.6189 gBDNF; r = -0.3554, p = 0.3806 gPBS) or 10 in 

either the grafted BDNF- or grafted PBS-infused animals (week 10 data not shown). 

287 

 
 
 
Figure 4.5: Exogenous BDNF infusion increases microglial (Iba1) expression in 
DA-grafted animals. 
(a) Confocal fluorescent micrograph and Imaris
and GFAP+ cells. Scale bar = 20 um. (b) Quantity of Iba1+ cells (volume um

3D image rendering highlighting Iba1+ 
3
) expressed  

TM 

288 

 
 
Figure 4.5 (cont’d) 
in the lesioned/grafted striatum of each treatment group. Ordinary one-way ANOVA with 
Šídák's multiple comparisons test; no significance between groups. (c) Quantity of Iba1+ 
3
)  normalized  to  the  number  of  grafted  TH+  neurons.  Mean  ±  SEM. 
cells  (volume  um
Unpaired t-tests. p = 0.0232. (d) Spearman correlation between quantity of Iba1+ cells 
3
(volume um
) normalized to the number of grafted TH+ neurons and total GID severity 
scores  at  week  5  post-engraftment.  Week  10  was  not  significant;  data  not  shown.  (e) 
3
Quantity of GFAP+ cells (volume um
) expressed in the lesioned/grafted striatum of each 
treatment group. Ordinary one-way ANOVA with Šídák's multiple comparisons test. p = 
3
0.0103  gBDNF  vs.  non-grafted  BDNF.  (f)  Quantity  of  GFAP+  cells  (volume  um
) 
normalized  to  the  number  of  grafted  TH+  neurons.  Statistics:  Mean  ±  SEM.  No 
significance.  (g)  Spearman  correlation  between  quantity  of  quantity  of  GFAP+  cells 
3
(volume um
) normalized to the number of grafted TH+ neurons and total GID severity 
scores  at  week  5  post-engraftment.  Week  10  was  not  significant;  data  not  shown.  (h) 
Serum concentration of IL-6 (pg/mL) from each treatment group. Statistics: Mean ± SEM. 
Non-parametric  Kruskal-Wallis  with  Dunn’s  multiple  comparisons  test.  (i)  Spearman 
correlation between serum concentration of IL-6 (pg/mL) and GID severity scores at week 
5  post-engraftment.  Week  10  was  also  not  significant.  Data  not  shown. Abbreviations: 
Iba1 = ionized calcium-binding adaptor molecule 1, GFAP = glial fibrillary acidic protein, 
IL-6 = interleukin-6.  

289 

 
 
 
 
 
DISCUSSION 

While neural transplantation does not offer a “cure” for PD, it does offer a 

promising non-pharmacological alternative to the therapies currently prescribed for PD. 

In both preclinical and clinical settings, the past two decades have seen rigorous 

research in neural grafting, taking strides to optimize patient selection (e.g., age, 

disease severity) and transplantation methods (e.g., cell source, preparation) (Barker et 

al., 2024). Despite refinement, many obstacles still exist with GID continuing to be a 

prominent, detrimental side effect. The lack of underlying mechanisms responsible for 

GID has generated a large gap in our understanding of how to make cell therapy a 

viable therapeutic option. In order to move forward, it will be imperative to harness the 

benefit while preventing the side effect of GID to fully optimize cell transplantation as a 

therapeutic for PD.  

One of the major focus areas in our laboratory involves striving to understand 

factors linked to GID in parkinsonian rats that receive eVM DA grafts. More recently, we 

began investigating GID in the context of the rs6265 SNP, testing the hypothesis that 

this SNP is an unrecognized contributor to the development of this side effect in a 

subpopulation of PD patients who received embryonic DA neuron grafts. Using the 

novel CRISPR knock-in rat model of the rs6265 BDNF SNP, we indeed demonstrated 

that parkinsonian rats homozygous for rs6265 (i.e., Met/Met) engrafted with WT DA 

neurons uniquely developed GID behavior compared to their WT counterparts engrafted 

with the same DA neurons (Mercado et al., 2021). We have also demonstrated, for the 

first time, that DA grafts exhibit neurochemical evidence of DA/glutamate co-

transmission evidenced by the expression of VGLUT2mRNA and protein co-localized 

290 

 
inside TH+ neuronal fibers (Mercado et al., 2021). Compellingly, only in the Met/Met 

hosts was VGLUT2 expression significantly correlated to GID behavior. As previously 

suggested (Kordower et al., 1996), our continuing research indicates that grafted eVM 

DA neurons maintain an immature phenotype (i.e., VGLUT2; (El Mestikawy et al., 

2011)), establishing asymmetric (presumed) glutamatergic synapses onto MSNs 

(Mercado et al., 2021; Soderstrom et al., 2008). These ultrastructurally-defined 

asymmetric synapses formed by grafted DA neurons positively correlated with an 

increase in GID (Soderstrom et al., 2008).  

Based on these compelling data pointing to improper DA-glutamate 

circuitry/wiring as a potential underlying mechanism of GID, we hypothesized that the 

rs6265 (Met/Met) host environment, due to a decrease in BDNF release, prevents 

proper graft maturation and permits synaptic miswiring of the transplanted DA neurons, 

thus giving rise to GID induction. Without sufficient BDNF, a protein critical for synaptic 

formation and dendritic spine formation (Gonzalez et al., 2016; Hyman et al., 1991; 

Kowiański et al., 2018; Lai & Ip, 2013; Park & Poo, 2013; Sasi et al., 2017; Zagrebelsky 

et al., 2020), the grafted neurons may not be able to form proper connections with the 

host MSNs. Consequently, within this current study, we predicted that, if we could 

“replenish” the deficient BDNF, grafted DA neuron maturation and proper graft-host 

integration would occur, preventing aberrant miswiring and behavior side effects (i.e., 

GID development).  

Strikingly, BDNF infusion into Met/Met parkinsonian rats engrafted with WT DA 

neurons exhibited significantly higher GID behavior compared to DA-grafted vehicle 

PBS-infused control animals. Further, not only was this demonstrated behaviorally, 

291 

 
BDNF infusion was also unsuccessful in allowing for maturation of the grafted DA 

neurons, evidenced by remaining expression of VGLUT2 protein inside TH+ neurites. 

Although VGLUT2 expression was no longer correlated with GID behavior in these DA-

grafted parkinsonian rats, it is probable that BDNF is not the sole factor required for full 

maturation of DA neurons transplanted into the mature adult parkinsonian striatum. 

Moreover, there is the possibility that four weeks of infusion was insufficient to induce 

full gestational maturation of these embryonic neurons placed into the adult striatum. 

These disparate findings collectively indicate that factors in addition to, or distinct from, 

BDNF and/or VGLUT2 expression contribute to GID induction in Met/Met parkinsonian 

animals.  

Despite a significant difference in GID behavior between infusion groups in this 

last study, no statistical differences were found in graft-mediated reduction in LID 

behavior. Interestingly, while grafted rs6265 Met/Met animals seem to uniquely develop 

GID behavior, this genotype type has conversely demonstrated enhanced behavioral 

recovery (i.e., LID amelioration) compared to their grafted WT counterparts in our 

previous study (Mercado et al., 2021). In the current experiment, we hypothesized that 

the DA-grafted BDNF-infused animals would have an even greater/faster amelioration 

of LID behavior than the DA-grafted PBS-infused animals. While this hypothesis was 

somewhat accurate, at the conclusion of the study, reduction in LID scores of both 

grafted groups were statistically similar, ultimately indicating that BDNF treatment did 

not effectively impact functional recovery in the paradigm employed.  

Because BDNF is known to induce neurite outgrowth in cultured neurons (Barde 

et al., 1982; Kellner et al., 2014) and in vivo ((Yurek et al., 1996; Yurek, 1998; J. Zhang 

292 

 
et al., 2011), we hypothesized that the grafts that received BDNF infusion would exhibit 

enhanced neurite outgrowth compared to grafts that received only PBS infusion. 

Indeed, the grafted BDNF-infused animals demonstrated significantly increased 

average neurite outgrowth, both in the proximal and distal regions of the graft, 

compared to the grafted PBS-infused animals. Moreover, in the distal region of the graft 

in these grafted BDNF-infused animals, the dorsolateral neurite outgrowth was 

remarkably higher than the neurite outgrowth in the same area of the grafted PBS-

infused animals. Additionally, these findings also confirm what has been shown with 

exogenous BDNF infusion in grafted parkinsonian rats in (Yurek, 1998; Yurek et al., 

1996). Because the cannula was placed directly dorsal to the graft in each animal 

subject to infused BDNF, it is understandable that the dorsolateral region in the grafted-

BDNF exhibited the most neurite outgrowth compared to grafted PBS-infused animals. 

We can infer that this increase in neurite outgrowth may have had a positive impact on 

the slight enhancement of graft efficacy in the grafted BDNF-infused animals, but we 

cannot yet definitively ascertain whether this increase in neurite outgrowth influences 

GID behavior. It could be postulated that increased neurite outgrowth leads to increased 

asymmetric synaptic connections, and therefore GID behavior; however, further 

investigation is warranted.  

 To further explore potential mechanisms of GID in the Met/Met parkinsonian 

animals and to understand how BDNF infusion could induce more severe GID, I utilized 

amphetamine-mediated rotational behavior and postmortem immunohistochemical 

expression of the DAT, VMAT2, and VGLUT2 proteins to help define the mystery of GID 

induction. 

293 

 
Amphetamine-mediated rotations, my secondary readout of graft function, does 

not necessarily determine graft size or extent of reinnervation (Björklund & Lindvall, 

2017); however, it can assess whether the transplanted graft is functioning properly. A 

unilaterally lesioned rat with no grafted DA neurons will rotate ipsilateral to the lesioned 

hemisphere upon amphetamine administration as detailed earlier (Dunnett & Torres, 

2011). After receiving a DA graft, the parkinsonian rat should no longer rotate if the graft 

is balances the amount of DA between the intact and lesioned striatal hemispheres. In 

this way, I used amphetamine-mediated rotations to indirectly assess DA release from 

the DA grafts in the presence or absence of BDNF supplementation. While the non-

grafted BDNF- or PBS-infused parkinsonian rats maintained a high level of ipsilateral 

rotations indicative of their lesioned status, the DA-grafted PBS-infused rats showed a 

normalization of rotational asymmetry. Compellingly, DA-grafted BDNF-infused animals 

rotated contralaterally to the lesioned hemisphere, suggesting that these grafted 

neurons were producing excess DA upon amphetamine administration in comparison to 

their vehicle-control counterparts. Curiously, rotations were not correlated to GID 

behavior. This functional measure of DA release provides insight into the consequences 

of BDNF administration and how it relates to one underlying mechanism of GID 

behavior (i.e., excess DA release).  

Due to the increase in contralateral rotations in the grafted BDNF-infused 

animals and the functional confirmation that these grafts are releasing more DA than 

observed in the DA-grafted PBS-infused group, I next analyzed the expression of DAT 

to confirm or refute the hypothesis that, if BDNF was promoting increased DA release, 

there would be increased DAT expression in the DA-grafted BDNF-infused animals 

294 

 
which would be significantly correlated to GID behavior. Indeed, the data demonstrate 

that DAT expression was significantly higher in the DA-grafted BDNF-infused animals 

compared to the DA-grafted PBS-infused animals. Importantly, the increase in DAT was 

also significantly correlated with GID scores in the DA-grafted BDNF-infused animals, 

supporting the association between GID behavior and DA release. Also at week 5, we 

compared DAT:TH intensity/um2 with net ipsilateral amphetamine-induced rotations. 

Further examining the relationship between DAT fluorescent intensity or TH intensity, 

only DAT and net ipsilateral rotations were correlated, confirming that DAT expression 

likely is related to functional DA release mediated by amphetamine. Thus, DA release 

continues to be a promising mechanism responsible for GID. It is also important to note 

that DAT function, not just expression (Bosse et al., 2012), could be altered as well, but 

further research would be required to evaluate this. 

It is not surprising that BDNF administration seemingly promotes DA release. 

Several groups have demonstrated a relationship between BDNF and DA, showing that 

BDNF plays a critical role in DA neurotransmission. For example, Blochl and colleagues 

demonstrated enhanced depolarization and basal DA release upon BDNF 

administration to E14 eVM cultured neurons (Blöchl & Sirrenberg, 1996). Similarly, 

BDNF stimulated DA uptake activity also in eVM cultured neurons ((Beck et al., 1993; 

Knüsel et al., 1991). Striatal in vivo infusions of BDNF increased electrical activity in rat 

midbrain DA neurons (Bosse et al., 2012; R. Y. Shen et al., 1994) and elevated activity-

dependent release of DA (Goggi et al., 2002) in both rat brain striatal slices and in the 

hippocampus (Paredes et al., 2007). Altar and colleagues, in contrast, did not see a 

change in striatal DA levels after two-week BDNF infusion to the SNpc in adult rats but 

295 

 
saw an increase in DA metabolite concentrations, indicating increased DA turnover, 

more so after amphetamine administration (Altar et al., 1992). Based on the evidence 

that BDNF administration evokes DA release, and because our study is consistent with 

these findings, I explored how this could mechanistically be related to GID induction. 

This exploration led me to a possible connection between DA release and glutamate co-

transmission. 

For over 20 years, it has been known that DA neurons have the potential to co-

transmit both DA and glutamate neurotransmitters. Yet, the functional significance and 

benefit behind this phenomenon remains uncertain. A number of laboratories have 

established that a subpopulation of DA neurons co-express/co-release DA and 

glutamate, marked by co-expression of either Vglut2 mRNA or VGLUT2 protein 

(Bérubé‐Carrière et al., 2009; Buck et al., 2022; Dal Bo et al., 2004; Fortin et al., 2019; 

Mingote et al., 2019; Root et al., 2016; T. Shen et al., 2018; Sulzer et al., 1998; Trudeau 

et al., 2014). Most have demonstrated that this subset of DA+/glutamate+ neurons are 

localized to the VTA, and only a small subset of these neurons project from the SNpc to 

the dorsal striatum (Buck et al., 2022; Eskenazi et al., 2021; Kawano et al., 2006). 

Behaviorally, VGLUT2 knock-out (KO) in DA neurons diminished neurochemical 

responses of mice to methamphetamine (H. Shen et al., 2021) and reduced locomotor 

response to cocaine (Hnasko et al., 2010), both of which are DA-releasing 

pharmacological agents. Furthermore, Hnasko and colleagues also demonstrated both 

decreased glutamate and DA release from ventral striatum slice cultures of VGLUT2 KO 

DA neurons (Hnasko et al., 2010), expressive of an important function of dual-release of 

these two neurotransmitters.  

296 

 
The most prominent theory that has been increasingly recognized as a logical 

functional explanation of DA/glutamate co-transmission, and the findings above in 

VGLUT2 KO DA neurons, is vesicular synergy (Figure 4.6). Vesicular synergy is a 

process that leads to enhanced loading of a primary neurotransmitter into secretory 

vesicles (for review (El Mestikawy et al., 2011)). For instance, it is well known that, with 

VGLUT3 and vesicular acetylcholine transporter (VAChT) on the same vesicle in 

cholinergic neurons, enhanced packaging of acetylcholine occurs (Gras et al., 2008). 

Although well established in this system, other systems such as dopaminergic neurons, 

GABA neurons, etcetera, remain relatively unexplored. In the dopaminergic system, the 

hypothesis of vesicular synergy suggests that the presence of VGLUT2 on the same 

vesicle as VMAT2 enhances the loading of DA, leading to increased DA release (Aguilar 

et al., 2017; Hnasko et al., 2010; H. Shen et al., 2018). Particularly, glutamate would 

enter through VGLUT2, increase the chemical gradient (Trudeau et al., 2014), and 

acidify the inside of the synaptic vesicle. Aguilar and colleagues has confirmed this, 

showing the hyperacidification of DA vesicles in a VGLUT2-dependent manner in mice 

(Aguilar et al., 2017). The increase in the chemical gradient promotes increased loading 

of DA through VMAT2, increasing the concentration and release of DA (Figure 4.6bc) 

(Eskenazi et al., 2021). To date, whether VMAT2 and VGLUT2 are on the same vesicle 

remains controversial (Aguilar et al., 2017; S. Zhang et al., 2015). One study has shown 

that a population of TH+/VGLUT2+ neurons in the VTA contain VMAT2 using PCR (Li et 

al., 2013), and another has shown co-immunoprecipitation of VMAT2 and VGLUT2 in a 

population of striatal synaptic vesicles (Hnasko et al., 2010; H. Shen et al., 2021; Silm et 

al., 2019). In contrast, Zhang and colleagues established that VMAT2 and VGLUT2 tend 

297 

 
to segregate into separate vesicles using immunolabeling, co-immunoprecipitation, and 

ultrastructural analysis in the adult nucleus accumbens (S. Zhang et al., 2015).  

Vesicular synergy and its potential for increased DA packaging and release 

would be an entirely novel explanation that takes into account both DA/glutamate co-

transmission (i.e., VGLUT2) and the DA release correlation we have demonstrated in 

our previous study (Mercado et al., 2021) and in my thesis studies, respectively. For 

confirmation, I endeavored to investigate whether I could find any evidence of VMAT2 

and VGLUT2 co-localization in the TH+ grafted neurons in the BDNF-infused Met/Met 

rats and whether any association with GID existed. I was able to demonstrate using 

triple-label immunohistochemistry, confocal microscopy, and the Imaris imaging 

software the existence of VMAT2/VGLUT2 (presumed) co-localizations in the TH+ 

neurons of DA-grafted Met/Met BDNF-infused rats that demonstrated a slight increase 

in number compared to DA-grafted PBS-infused rats. It is noteworthy that this presumed 

co-localization of VMAT2/VGLUT2 in this treatment group was strongly correlated with 

GID behavior, demonstrating additional favorable evidence that GID behavior may 

indeed be caused by increased DA release mediated by vesicular synergy within grafted 

DA neurons. Nevertheless, additional studies are required to determine undoubtedly 

that these proteins are within the same synaptic vesicle. 

298 

 
 
 
Figure 4.6: Schematic diagram depicting the proposed mechanism of vesicular 
synergy. 
(a) Levodopa taken up into the dopaminergic neuron and converted to dopamine via 
aromatic amino acid decarboxylase (AADC). Normal packaging of dopamine occurs 
here; DA is released into the synapse and activates both D1 and D2 receptors on the 
post-synaptic membrane of MSNs. (b) At baseline, VMAT2 on a DA synaptic vesicle will 
exchange 2 hydrogen ions for one molecule of DA to achieve sufficient DA uptake and 
release. (c) In synaptic vesicles that co-express VMAT2 and VGLUT2, VGLUT2 will 
transport one chlorine and one phosphate ion, acidifying the inside of the vesicle, 
thereby increasing the concentration gradient, and ultimately resulting in the uptake of 
an increased amount of DA molecules via VMAT2. (d) Subsequently, the uptake of more 
dopamine will lead to the increased dopamine release from these VMAT2/VGLUT2 
vesicles. Adapted from (Eskenazi et al., 2021).   

299 

 
 
 
 
Although I recognize that there has been conflicting evidence of both the co-

localization of DA/glutamate co-release in the dorsal striatum (responsible for motor 

behavior) and the co-localization of VMAT2/VGLUT2 on the same vesicle, it is not yet 

possible to exclude the possibility that both exist in the context of neural grafting in our 

experiments. No other group has investigated these phenomena in a grafted rs6265 

Met/Met parkinsonian rat, or in other models of DA neuron grafting, to the best of my 

knowledge. Our behavioral results and postmortem analyses are promising evidence in 

support of the theory of vesicular synergy, and vesicular synergy offers a logical 

mechanism responsible for GID behavior, at least in the context of this study. Future 

research further examining the potential for VMAT2/VGLUT2 co-localization are 

warranted and could offer new avenues for therapeutic development to prevent GID in 

patients.  

In addition to evidence of excess DA release and DA/glutamate co-transmission, 

past experiments have also revealed a role of the immune system in GID behavior, 

including a study conducted by our group (see Soderstrom et al., 2008). Grafted 

parkinsonian animals exposed to immune activation exhibited increased GID severity 

compared to non-challenged rats (Soderstrom et al., 2008). In the clinic, patients who 

underwent withdrawal of immunosuppression (Hagell & Cenci, 2005; Olanow et al., 

2003) or did not receive immunosuppression (Freed et al., 2001) developed GID 

behavior. Therefore, we considered the presence of the immune markers Iba1 

(microglia) and GFAP (astrocytes). Grafted BDNF-infused parkinsonian rats showed an 

increase in expression of Iba1 in comparison to grafted PBS-infused animals but no 

differences in GFAP expression. Notably, there was no correlation between Iba1 

300 

 
expression and GID in these animals. Nevertheless, this does not mean the immune 

system does not play a role in GID development. Future studies warrant analysis of 

additional immune markers and could also take into account activated versus 

inactivated microglia and morphology. Furthermore, it would have been advantageous 

to examine postmortem tissue immediately following cessation of BDNF infusion instead 

of at the end of 10 weeks post-engraftment, although resources were not available to 

investigate this for my thesis studies. GID severity differences were more prominent 

between treatment groups at week five post-engraftment, and acute effects of BDNF on 

these immune markers could have been more apparent at this timepoint. Lastly, studies 

directly assessing the association between GID development and immune suppression 

are needed to definitively confirm the role of the immune system, and more importantly, 

how to abate these factors to allow neural grafting to become a more uniformly effective 

therapy option. 

Our current study has demonstrated that exogenous BDNF treatment does not 

induce maturation of DA neuron transplants and would not be a safe and/or efficacious 

solution in the clinic to prevent GID as a side effect for grafted parkinsonian patients. In 

spite of this, we did, however, present evidence that confirms the clinical GID 

pharmacotherapy (i.e., buspirone) and offers great promise for the role of excess DA 

release and/or vesicular synergy underlying GID behavior. Collectively, these results 

provide a foundation for an abundance of future investigations. Furthermore, as the 

colocalization of VMAT2/VGLUT2 can only be presumed, additional methods (e.g., 

proximity ligation assays, ultrastructural analysis) will be necessary to prove, without a 

doubt, that these proteins are indeed co-localized together on the same synaptic 

301 

 
vesicle. Keeping the current precision-medicine climate in mind, these experiments, 

along with our other studies, continue to provide a convincing argument for genotyping 

patients prior to their participation in cell transplantation trials for PD. 

302 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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CHAPTER 5: FUTURE DIRECTIONS AND CONCLUDING REMARKS 

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Parkinson’s disease (PD) is a relentlessly progressive neurodegenerative 

disorder that continues to negatively affect society. As we continue to study PD, the 

leading consensus is that PD is not a unitary disease entity; it is instead a multifaceted 

clinical syndrome with complex, heterogeneous etiologies. In this way, safe, efficacious 

treatments have been considerably difficult to develop. Indeed, as has been discussed 

at length in this thesis, several available therapeutic options are prescribed to treat PD, 

yet patient responsiveness is not uniform (Bove & Calabresi, 2022; Fabbri et al., 2016; 

Varanese et al., 2010), and even the best therapies have incomplete and/or limited 

lasting benefit. Most notably, with levodopa treatment, significant heterogeneity remains, 

both in clinical benefit and in the development of levodopa-induced dyskinesia (LID). As 

a beacon of hope, various experimental procedures are being examined as potential 

alternatives to the current dopamine (DA)-replacement strategies. Some examples 

include developing extended-release agents to achieve long-acting levodopa release 

and creating gene-therapy agents that target α-synuclein pathology. Arguably one of the 

most promising is the focus of my thesis work—regenerative neural cell replacement 

therapy. While cell therapy is not, nor will ever be, considered a “cure” for PD, scientists 

and clinicians have endeavored to optimize neural transplantation as a one-time 

procedure that will offer symptomatic relief for individuals with PD for decades to come 

(Barker et al., 2024). 

In the following sections, I will discuss the key findings from my dissertation 

research and how these findings positively contribute to the field of neural 

transplantation for PD. Moreover, I will discuss limitations and potential caveats of my 

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studies, and I will share additional insight into how evidence collected here provides a 

strong foundation for the continuation of this research.  

USING PRECISION MEDICINE TO GARNER MECHANISTIC INSIGHTS INTO GID 

BEHAVIOR 

To date, the underlying cause of the aberrant side effect, graft-induced 

dyskinesia (GID), in response to neural transplantation of replacement DA neurons into 

the parkinsonian striatum remains elusive. Upon the conclusion of two clinical grafting 

trials funded by the NIH in the early 2000s, clinicians discovered this novel dyskinetic 

behavior that manifested only in subpopulation of individuals who received primary 

embryonic ventral mesencephalic (eVM) DA grafts (Freed et al., 2001; Olanow et al., 

2003). Consequently, a worldwide mortarium was enacted following these trials as well 

as a clinical trial is Sweden (Hagell et al., 2002), halting all clinical grafting trial for PD 

(Hagell & Cenci, 2005). Now, after rigorous preclinical studies and re-evaluation of 

clinical studies, clinical grafting trials are scheduled or currently ongoing (see Table 1.1 

in Chapter 1 for a comprehensive list). Therefore, our research group has posited that, 

for cell transplantation to be an optimal therapeutic for patients with PD, we must 

investigate and elucidate the heterogeneity, namely GID development, in this 

subpopulation of patients.  

As discussed in previous chapters, we have been studying the common human 

single nucleotide polymorphism (SNP), rs6265, as a potential underlying genetic risk 

factor for the development of GID behavior due to its resultant decrease in activity-

dependent release of BDNF (Egan et al., 2003). Using a CRISPR knock-in parkinsonian 

rat model of the rs6265 SNP (Met/Met), my predecessor, Dr. Natosha Mercado, 

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demonstrated that Met/Met parkinsonian rats paradoxically exhibited enhanced 

functional recovery (i.e., earlier/more robust amelioration of LID behavior) and neurite 

outgrowth following primary eVM transplantation from wild-type (WT; Val/Val) donor 

neurons; however, these animals uniquely developed significant GID behavior 

compared to their WT counterparts (Mercado et al., 2021).  

To complement her findings, I was able to demonstrate that, when additional 

host/donor combinations were studied (i.e., WT and Met/Met hosts engrafted with WT or 

Met/Met donor neurons), the homozygous rs6265 Met/Met genotype retained its 

beneficial functional action compared to the WT genotype, shown by an earlier 

amelioration of LID behavior (Chapter 3). Strikingly, however, I found that Met/Met rats 

engrafted with WT DA neurons were the only host/donor combination to exhibit 

significantly meaningful GID. Based on the similarities in GID induction in this genotypic 

host/donor combination between my study and Dr. Mercado’s, I endeavored to 

investigate underlying mechanisms that may be responsible for this aberrant DA-graft-

mediated behavior.  

In both my in vivo studies, the data suggests that an increase in DA release (i.e., 

DAT expression) was prevalent and positively associated with GID behavior, which 

showed strong statistical significance in the exogenous BDNF supplementation study 

(see Chapter 4). I additionally demonstrated that, inside the grafted DA neurons of the 

BDNF-infused animals, there is (presumed) co-localization of vesicular monoamine 

transporter 2 (VMAT2) and vesicular glutamate transporter 2 (VGLUT2) which 

correlated strongly to GID behavior. This novel finding, again, is suggestive of vesicular 

synergy, which provides a logical explanation for how excess DA release and 

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DA/glutamate co-transmission could both be implicated in GID behavior. This evidence 

does not stand alone: other evidence in favor of excess DA release and/or 

DA/glutamate co-transmission in GID behavior has been collected clinically and 

preclinically. Indeed, fluorodopa positron emission topography (FDOPA PET) scans in 

graft recipients indicated that increases of DA were apparent in patients who exhibited 

GID compared to those who were GID-negative (Ma et al., 2002) (see Chapter 1: 

“Uneven DA reinnervation/DA release”). Shin and colleagues demonstrated that 

pharmacological blockade of D2 receptors using a DA antagonist, buspirone, resulted in 

significantly diminished GID behavior (Shin et al., 2012). Additionally, in the same 

preclinical rat model as my studies, Mercado and colleagues reported an upregulation 

of Drd2 mRNA in the host MSNs of Met/Met rats engrafted with WT DA neurons which 

correlated with GID (Mercado et al., 2024). Lastly, for DA/glutamate co-transmission, 

Met/Met rats exhibited VGLUT2 expression that correlated strongly with GID behavior 

(Mercado et al., 2021). These previous analyses, along with the current evidence I have 

collected, strongly corroborate that there is a link between excess DA release and 

DA/glutamate co-transmission as an underlying mechanism of GID behavior. To the 

best of my knowledge, this is the first study to discover (presumed) co-localization of 

VMAT2 and VGLUT2 in grafted eVM neurons and the theory of vesicular synergy in the 

expression of GID, and therefore, this is an innovative avenue that should be examined 

further.  

While we had hypothesized that BDNF-mediated maturation may be a way to 

prevent GID, it is also important to note that, because exogenous BDNF administration 

exacerbated GID behavior in grafted Met/Met parkinsonian rats, this does not appear to 

318 

 
be a safe or effective option to treat GID in grafted patients with PD. For years, BDNF 

administration has been considered a potential neuroprotective agent that could prevent 

the degeneration of nigrostriatal DA neurons (Nagahara & Tuszynski, 2011). Although 

not yet tested clinically in PD, preclinical studies of neurodegenerative and psychiatric 

disorders have utilized intrastriatal injections of viral vectors with the BDNF gene to 

increase BDNF protein production (Chen et al., 2020; Kells et al., 2004) (see Chapter 

2). Engineered fibroblasts that produce BDNF have also been transplanted into the 

brain to study its therapeutic potential (Kells et al., 2004; Levivier et al., 1995). Despite 

some promising reports, it is difficult to tightly regulate gene therapy for BDNF, and 

overproduction can negatively affect host circuitry (Yeom et al., 2016; Zuccato & 

Cattaneo, 2009) (see (Szarowicz et al., 2022) for review). Therefore, until these BDNF 

administration therapies are optimized, and based on the results from my studies, 

supplementing with BDNF cannot be recommended, especially in this context of clinical 

grafting in PD.  

After reviewing the clues assembled from my studies regarding GID behavior, 

along with several other comparable experiments, it is clear there is no “one-size-fits-all” 

approach to effectively treat PD. Ultimately, the conclusions regarding the rs6265 SNP 

provide a compelling argument for implementing a precision-medicine-based approach 

in neural transplantation for PD. Accordingly, Figure 5.1 illustrates a proposed 

precision-based therapeutic approach to prevent and/or treat GID in the context of 

neural transplantation based on the findings discussed above. Specifically, it is 

reasonable to recommend that both the recipient and donor neurons are genotyped for 

the rs6265 SNP as a way of preventing GID development following DA neuron 

319 

 
transplantation (Figure 5.1a). Alternatively, in patients who have already received DA 

transplants, and subsequently developed GIDs, various tailored therapeutics, once 

discovered, could be administered to target the underlying mechanisms, therefore 

preventing GID occurrence (Figure 5.1b).  

Figure 5.1: A possible precision-medicine-based therapeutic approach to prevent 
and/or treat GID behavior prior or following DA cell transplantation. 
(a) Prior to grafting, genotyping the human patient and the donor neurons for the rs6265 
SNP, is a recommended precision-based approach aimed at preventing the development 
of GID behavior. Since our previous (Mercado et al., 2021, 2024) and current studies have 
demonstrated  that  the  homozygous  Met/Met  host parkinsonian  rats  engrafted  with  WT 
DA  neurons  uniquely develop  GID  behavior,  preventing  this host/donor  transplantation 
combination would help avoid GID induction in grafted patients with PD. (b) Following cell 
transplantation, several mechanisms that could underlie GID behavior are proposed here 
as  targets.  For  example,  if  excess  DA  release  is  responsible  for  GID  behavior,  DA 
antagonists  such  as  buspirone  can  be  administered  to  prevent  potential  aberrant  GID 
behavior. With our studies, we have ruled out the administration of exogenous BDNF as 
it  was  shown  to  exacerbate  GID  behavior  in  Met/Met  host  rats  engrafted  with  WT  DA 
neurons (see Chapter 4).  

320 

 
 
THE FUNCTIONAL BENEFIT OF rs6265 AND A POTENTIAL ROLE FOR THE BDNF 

PRO-PEPTIDE 

Although not at the forefront of my thesis studies, I did confirm that the 

homozygous rs6265 Met/Met genotype confers a degree of functional benefit and 

recovery following cell transplantation when compared to the WT genotype. Specifically, 

regardless of the presence of rs6265 in the host or donor neurons, animal groups with 

the Met/Met genotype demonstrated enhanced behavioral efficacy with earlier 

amelioration of LID behavior along with improvement of amphetamine-mediated 

rotational asymmetry compared to the WT hosts engrafted with WT DA neurons (see 

Chapter 3). Findings gained from my studies reinforce what was reported in (Mercado et 

al., 2021): Met/Met parkinsonian hosts exhibited enhanced graft-derived efficacy and 

increased neurite outgrowth in comparison to WT hosts (Mercado et al., 2021). 

Furthermore, a potential benefit of the Met allele (both heterozygous and homozygous) 

has been shown in other neurological conditions including traumatic brain injury (TBI) 

(Barbey et al., 2014; Finan et al., 2018; Krueger et al., 2011), stroke (Qin et al., 2014), 

multiple sclerosis (MS) (Zivadinov et al., 2007), Alzheimer’s disease (AD) (Voineskos et 

al., 2011), and peripheral nerve injury (McGregor et al., 2019) (see Chapter 3: 

Discussion). Evidently, these reports contradict the historical paradigm that the Met 

allele is solely a “risk” allele. Instead, they indicate that the Met allele has, at least to 

some extent, some evolutionary benefit that explains its relatively high prevalence 

(approximately 20%) (Mercado et al., 2021; Petryshen et al., 2010; Tsai, 2018) in the 

general human population.  

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The rs6265 SNP is found within the prodomain/pro-peptide region of the BDNF 

gene, and therefore, this may suggest an important role for the BDNF Met pro-peptide 

in neuroregeneration based on the unexpected benefit addressed above. 

Encouragingly, researchers have recently become aware that the BDNF pro-peptide 

appears to act an independent and functional ligand similar to that of mature and 

proBDNF (Anastasia et al., 2013; Kojima & Mizui, 2017; Mizui et al., 2017). For 

example, several research studies have investigated the expression levels and 

differential function of both the WT and Met BDNF pro-peptides, albeit mostly in the 

region of the hippocampus (see Table 5.1 for a comprehensive list of experiments). In 

the context of our parkinsonian rat model, I hypothesized that the BDNF Met pro-

peptide may be responsible for permitting enhanced neurite outgrowth of transplanted 

DA neurons demonstrated in (Mercado et al., 2021) and enhanced functional recovery 

((Mercado et al., 2021); Chapter 3 of my studies).  

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Table 5.1: Evidence of varied BDNF pro-peptide activity associated with rs6265 
SNP expression. 
Abbreviations: HC: hippocampus, mPFC: medial pre-frontal cortex, CSF: cerebral spinal 
fluid, MDD: major depressive disorder, SCZ: schizophrenia, B6: C57BL/6, NAc: nucleus 
accumbens, BD: bipolar disorder, E: embryonic, DIV: Day in vitro, SD: Sprague Dawley, 
AD: Alzheimer’s  disease,  CBM:  cerebellum,  PC:  parietal  cortex,  P:  postnatal. Adapted 
from (Szarowicz et al., 2022). 

323 

Study  Subjects/Model Region/Source Effect Dieni et al. (2012)    C57BL/6, Bdnf-Myc, cbdnf ko, Bsn mutant mice all 8 weeks of age. Hippocampus  Mature BDNF and the BDNF pro-peptide are stored at equimolar ratios in large dense core vesicles in presynaptic terminals of excitatory neurons. Anastasia et al. (2013) Cultures prepared from E18 BDNFVal/Val and BDNFMet/Met knock-in mice   Primary neurons were isolated from E15 C57BL/6 mouse embryos. Hippocampal-cortical neurons     Hippocampus In hippocampal-cortical neurons, secreted levels of Met prodomain was significantly lower compared to Val prodomain secretion.  In hippocampal neurons, growth cone retraction was induced by Met prodomain application in p75+ cells; Val prodomain was inactive. Met prodomain only interacted with SorCS2 receptor. Lim et al. (2015) SH-SY5Y neuroblastoma cells   Extracts from post-mortem tissue (AD patients) Hippocampus In culture, application of the Met prodomain negatively affected cell viability only in the presence of Aβ; Val prodomain had no effect.  Levels of pro-peptide were 16-fold higher in AD patients and correlated with Aβ accumulation. Mizui et al. (2015)   Slices prepared from 3–4-week-old C57BL/6 and Bdnf KO mice.    DIV21 cultures prepared from E18 Wistar rats. Hippocampal tissue slices      Hippocampus Application of the Val pro-peptide facilitated LTD in hippocampal slices and required the activation of GluN2B-containing NMDA receptors.  In cultured neurons, Val pro-peptide also induced endocytosis of AMPA receptors.  In cultured neurons, the presence of the Val66Met SNP in the pro-peptide inhibited LTD.    
 
Table 5.1 (cont’d)  

324 

Study  Subjects/Model Region/Source Effect Guo et al. (2016)  DIV16 rat neuronal cultures electroporated with plasmid-expressing eGFP. Hippocampus  Val prodomain application reduced spine density and increased spine length.  Val prodomain increased caspase-3 activity and mitochondria elongation. *Met prodomain was not studied. Yang et al. (2016) 7-week-old male Sprague Dawley rats of learned helplessness (LH) model of depression (WT and Bdnf KO). Medial prefrontal cortex (mPFC), CA3 and dentate gyrus of hippocampus, nucleus accumbens. Significantly higher expression of BDNF pro-peptide in mPFC and CA3 regions of LH rats compared to controls.   Significantly lower expression of BDNF pro-peptide in nucleus accumbens and dentate gyrus compared to controls. Uegaki et al. (2017) BIAcore sensor chip and recombinant human BDNF protein  Slices prepared from male C57BL/6J mice (3-4-weeks-old)       Hippocampus Using BIAcore chip, the BDNF pro-peptide binds to mature BDNF with high affinity.  Using BIAcore chip, The Met pro-peptide is more stable in acidic and neutral pH environments compared to Val pro-peptide.  In hippocampal slices, pre-incubation of the Val pro-peptide reduced the ability of mBDNF to inhibit LTD. Yang et al. (2017)  Patients with MDD, SCZ, and bipolar disorder (BD) Postmortem samples of cerebellum, parietal cortex, liver, and spleen BDNF pro-peptide levels were significantly lower in the cerebellum and the spleen of MDD, SCZ, and BD patients compared to control groups.  BDNF pro-peptide levels were significantly higher in the parietal cortex of MDD, SCZ, and BD patients compared to control groups.  
 
Table 5.1 (cont’d)  

To test my hypothesis, I employed an in vitro cell culture method which involved 

plating WT E14 eVM DA neurons (same cell source as our grafts) and treating them 

with exogenous administration of the WT or Met pro-peptide (25 ng/mL based on 

(Anastasia et al., 2013)) once per hour for a total of 16 hours (DIV4-5). Cells were then 

fixed with 4% paraformaldehyde and fluorescently stained for tyrosine hydroxylase (TH) 

to identify DA-positive neurons. Strikingly, Met pro-peptide application increased both 

the number and volume of TH+ neurons (Figure 5.2), indicating that the BDNF Met pro-

peptide may have a positive impact on embryonic DA neurons, at least within this 

primary cell source. Further trials of this experimental design are warranted in order to 

325 

Study  Subjects/Model Region/Source Effect Giza et al. (2018)  DIV21 primary neurons prepared from C57BL/6 mice.     BDNFVal/Val, BDNFVal/Met, BDNFMet/Met P23-P60 male mice. Hippocampus (ventral CA1 neurons)      Hippocampus In culture, the Met prodomain decreased mushroom spines and reduced PSD95 density in p75+ and SorCS2+ cells; Val prodomain had no effect.   Increased freezing behavior/decreased fear extinction was demonstrated in Met-prodomain injected mice. Fewer spines were also found in Met-prodomain treated mice compared with the Val-prodomain injected mice. Mizui et al. (2019) Japanese patients with Major depressive disorder (MDD) or Schizophrenia (SCZ) Cerebral spinal fluid The ratio of BDNF pro-peptide to total protein in MDD patients was lower in males and not females compared to controls.  The ratio of BDNF pro-peptide to total protein was lower in SCZ patients, but it was not statistically significant.            
 
 
optimize the timing of application (e.g., continuous vs. per hour administration) and pro-

peptide concentration. Again, while this was an ancillary study of my thesis, these 

findings provide an exciting new path for future research on the paradoxical benefit of 

the Met-allele.  

a) 

b) 

Figure 5.2: Impact of the BDNF Met and WT pro-peptides on survival and volume 
(µm3) of TH+ DA neurons in cell culture. 
(a) Number of TH+ dopaminergic neurons following treatment of the BDNF Met or WT 
pro-peptide  and  their  respective  controls  (i.e.,  mature  BDNF  or  water  as  a  negative 
control). Statistics: Mean ± SEM, Ordinary one-way ANOVA with Tukey’s multiple  
comparisons,  p   =  0.0194  mBDNF vs.  Met  pro-peptide-treatment; p  = 0.0168  negative 
control vs.  Met  pro-peptide-treatment.  (b)  Average  volume  (µm3) of TH+  dopaminergic 
neurons following treatment of BDNF Met or WT pro-peptide and their respective controls. 
Statistics: Mean ± SEM, Ordinary one-way ANOVA with Tukey’s multiple comparisons, p 
= 0.0179 mBDNF vs. Met pro-peptide-treatment; p = 0.0115 negative control vs. Met pro-
peptide-treatment.  

LIMITATIONS AND ALTERNATIVE APPROACHES 

Two limitations of my studies include the exclusion of heterozygous rs6265 

(Val/Met) parkinsonian rats and the exclusion of female parkinsonian rats. These 

omissions were made for several practical reasons. In our first proof-of-concept 

experiment (Chapter 3), only WT and homozygous rs6265 (Met/Met) animals were 

studied in order to maximize the chances of observing any effect that might be 

326 

 
 
 
associated with the rs6265 SNP as the homozygous rs6265 genotype has the largest 

decrease in activity-dependent release of BDNF (Mercado et al., 2021). Additionally, 

because several host/donor combinations were being studied, including another 

genotypic profile would not be feasible to maintain as the total animals would be too 

numerous for proper behavioral analysis. The homozygous Met/Met genotype engrafted 

with WT DA neurons was employed for the second study (Chapter 4) as that was the 

only host/donor combination in our previous studies to develop significantly meaningful 

GID behavior. Similarly, only male rats were studied to maintain experimental feasibility. 

Because BDNF is well-known to interact with sex hormones (Chan & Ye, 2017; Wei et 

al., 2017), and rs6265 has been found to drive sex-specific susceptibility in various 

neurological disorders such as AD (Laing et al., 2012), MDD (Chagnon et al., 2015; 

Tsai, 2018), and schizophrenia (Chao et al., 2008; Suchanek et al., 2013; Yi et al., 2011) 

(see (Szarowicz et al., 2022)) for review), it would be highly relevant to conduct future 

studies repeated in females moving forward.  

We had originally planned to include only two grafted cohorts for the second 

experimental study with BDNF infusion (i.e., DA-grafted BDNF-infused and DA-grafted 

PBS-infused animals) (Chapter 4). In one cohort of animals, we had explored the idea 

of sacrificing immediately following osmotic minipump removal at the end of the four 

week infusion, and the other cohort was to be extended an additional six weeks after 

pump removal. However, due to unforeseen obstacles, we had to re-design our 

experiment: after purchasing timed-pregnant female rats for cell collection, the rats were 

not pregnant. Because we had already begun priming the pumps with BDNF and PBS, 

we made the decision to re-design the experiment to instead include “non-grafted” 

327 

 
BDNF- and PBS-infused animals and extend all animals to 10 weeks post-engraftment. 

In addition to this, we could have alternatively extended BDNF supplementation for a 

longer period; however, we were restricted by the cost of the BDNF protein (i.e., 

$50,000 for 8 mg). Despite these difficulties, the results obtained from these first proof-

of-concept studies are meaningful and provide a foundation for additional future 

research.   

For both in vivo experiments, I was only able to use indirect markers to determine 

whether there was evidence of differences in DA release (i.e., DAT expression) and 

immune activation (i.e., Iba1, GFAP). Although the markers I employed provide a 

sufficient starting point, neither directly demonstrate whether excess DA release or 

immune activation are underlying mechanisms of GID behavior. For instance, the DAT 

protein was immunohistochemically stained postmortem to indirectly assess DA release; 

however, DAT expression alone may not be affected in these animals. Accordingly, 

investigating the function of the receptor or other DA markers (e.g., D1/D2 receptors) 

would be a great addition to further assess whether DA release is indeed associated 

with GID in this model. Future experiments designed to directly measure DA release at 

the grafted DA neurons in the striatum would be important to collect definitive evidence. 

Such studies could involve in vivo voltammetry, which is further discussed below.  

Moreover, for immune markers, Iba1 and GFAP were chosen because both are 

pan markers commonly utilized to identify microglia and astrocytes, respectively. 

However, because they are solely pan markers, I was limited in realizing whether these 

immune cells are active or inactive as their presence alone does not necessarily reflect 

an increased activation of the immune system. Alternatively, other markers such as 

328 

 
MHC-II could be used, or the morphology of Iba1+ and GFAP+ cells in my studies could 

be analyzed in the future as another indication of activated vs. inactivated microglia or 

astrocytes, respectively.  

My data demonstrate the novel co-localization of VMAT2 and VGLUT2, which 

correlated significantly with GID behavior in DA-grafted BDNF-infused Met/Met 

parkinsonian rats. Again, to the best of my knowledge, this is the first time VMAT2 and 

VGLUT2 are shown to co-localize in grafted eVM neurons. However, as addressed in 

Chapter 4, the fluorescent immunohistochemistry approach I used for the analysis of 

VMAT2/VGLUT2 was limited as it could not visualize specific synaptic vesicles, which 

are only roughly 40 nm in diameter. Therefore, an additional approach to general 

immunohistochemical analysis is to employ electron microscopy or a proximity ligation 

assay (PLA), which is discussed in detail below. 

Lastly, to expand the translatability of my studies to the current clinical trials that 

are planned or ongoing, using another cell source other than primary DA neurons would 

be beneficial. For example, due to several ethical concerns and difficulty in obtaining 

sufficient quantities of embryonic cells, induced pluripotent stem cells (iPSCs) are a cell 

source that clinicians and researchers are shifting toward utilizing (Barker et al., 2024). 

In my experiments, we employed primary embryonic DA neurons because this is the 

only cell source that is currently known to induce substantial GID behavior. 

Nevertheless, alternative tests that employ iPSCs transplants are warranted to achieve 

an experimental design that is more translatable to human trials.  

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The Benefit of the Met allele and the BDNF Met Pro-peptide 

FUTURE DIRECTIONS 

As discussed previously in Limitations and Alternative Approaches, I conducted 

an in vitro cell culture experiment aimed at studying the differential impact of the BDNF 

WT vs. Met pro-peptide on neurite outgrowth in primary eVM DA neurons. In order to 

focus on the detriment, rather than the benefit, of the rs6265 Met/Met genotype in 

parkinsonian rats, I have not included these data in my dissertation. It is important to 

note, however, that our group submitted these cells to NanoString to be analyzed for 

differences in transcriptomic profiles between the WT- and Met-pro-peptide-treated 

cells, and we are currently in the process of analyzing the results. If certain genes 

related to neurite outgrowth are upregulated in the Met-pro-peptide-treated cells, these 

genes could potentially be targeted therapeutically to enhance the beneficial outcomes 

of neural transplantation in rs6265-carrying subjects. 

Directly measuring DA release 

Previous clinical trials have demonstrated promising evidence in favor of excess 

DA release underlying GID behavior. For instance, FDOPA PET signals were 

significantly higher in grafted patients who developed GID compared to the patients who 

did not (Ma et al., 2002) (see Chapter 1: Uneven DA reinnervation/DA release). Now, 

with our preclinical parkinsonian model, I have demonstrated indices of excess DA 

release in WT-grafted homozygous rs6265 (Met/Met) parkinsonian rats (i.e., increased 

DAT expression) (Chapter 3 and 4). Regrettably, however, both PET scans and DAT 

expression are not direct measures of DA release, so we can only infer that increased 

DA is associated with GID behavior. Therefore, future studies that directly assess DA 

330 

 
release from the graft are warranted. Accordingly, it would be advantageous to employ 

in vivo voltammetry, a technique that is commonly used to measure neurotransmitter 

release concomitantly with behavioral assessment. With this technique, we could be 

more confident whether DA release underlies GID behavior in this parkinsonian rs6265 

rat model.  

Co-localization of VMAT2/VGLUT2 and Vesicular Synergy 

It is likely that excess DA alone does not result in GID behavior, and thus, I 

investigated a possible connection between DA release and glutamate co-transmission 

based on previous findings that correlated VGLUT2 expression to GID in Met/Met hosts 

engrafted with WT DA neurons (Mercado et al., 2021). My results demonstrated that the 

number of (presumed) co-localized VMAT2/VGLUT2 was strongly, and significantly, 

correlated with GID behavior in WT DA-grafted BDNF-infused Met/Met parkinsonian rats 

(see Chapter 4), providing a compelling argument for the theory of vesicular synergy in 

these animals. However, VMAT2 and VGLUT2 are only presumed to be colocalized in 

the grafted DA neurons due to the limitations of magnification in my postmortem 

analyses. Thus, as a future direction, I propose employing a PLA assay that would aid in 

the definitive determination of whether VMAT2 and VGLUT2 are co-localized on the 

same synaptic vesicle. Using the PLA assay, the transporters will fluoresce if they are 

found within 40 nm of each other in the grafted DA neurons. Because there remains 

considerable contention as to whether VMAT2 and VGLUT2 are found on the same 

vesicle (e.g., (Aguilar et al., 2017; Zhang et al., 2015)), and as VMAT2/VGLUT2 have 

not been studied in eVM tissue or in the context of neural grafting, findings that 

demonstrate same vesicle colocalization could be groundbreaking.  

331 

 
A promising role for the immune system 

Although no exhibition associating the immune response to GID behavior was 

found in my studies, this does not exclude immune activation from potentially underlying 

GIDs, especially due to the limitations of immune marker analysis addressed in 

Limitations and Alternative Approaches. Indeed, clinical trials (Freed et al., 2001; 

Olanow et al., 2003) have demonstrated that, only after immunosuppression was 

discontinued, GIDs developed in DA-grafted individuals with PD. Our group has 

additionally confirmed that, in preclinical parkinsonian rat studies, GID severity was 

increased following immune challenge (Soderstrom et al., 2008). Future studies should 

directly investigate the role of immune activation in our rs6265 parkinsonian rat model to 

determine whether immune function correlates to GID behavior. In this way, 

immunosuppression agents (Figure 5.1b) could be given to DA-grafted WT and rs6265 

(Met/Met) parkinsonian rats; GID behavior and postmortem morphological changes of 

immune markers could then be assessed. Ultimately, this could be a promising potential 

therapeutic target aimed at the prevention of GID.  

Graft Location  

While not quantitative, I anecdotally observed that the location of the graft (e.g., 

dorsolateral vs. ventrolateral) influenced the development of GID behavior in both of my 

studies. For instance, in the host/donor combination study, I was able to qualitatively 

show that the homozygous rs6265 Met/Met hosts engrafted with WT DA neurons 

demonstrated a higher percentage of DA grafts placed in the dorsolateral region of the 

striatum and subsequently had a higher occurrence of significant GID induction 

compared to the other DA-grafted host/donor groups (see Figure 5.3). Likewise, in my 

332 

 
second experiment, based on the neurite outgrowth data (see Chapter 4), the higher 

neurite density of the graft was found in the dorsolateral region of the striatum in the 

DA-grafted BDNF-infused animals, the same group that exhibited significant GID 

behavior. Differential graft placement could also have been a contributor of GID in 

clinical trials. For example, in the Denver/Columbia trial (Freed et al., 2001), GID mainly 

affected the upper body, manifested primarily as dystonic movements, and increased 

FD uptake (PET) in the dorsal putamen. Conversely, in the Tampa/Mount Sinai trial 

(Olanow et al., 2003), GID developed largely in the lower extremities with more 

stereotypic movements, and FD uptake was increased in the ventral region of the 

putamen. These differences in GID suggest that variability is likely due to the differential 

placements of the DA graft (Steece-Collier et al., 2012).  

Since the dorsal and ventral striatum have differential functions, these findings 

are not entirely unexpected. Indeed, the dorsal striatum generally controls motor and 

cognitive function (Cataldi et al., 2022; Corbit et al., 2017; Haith & Krakauer, 2018), 

while the ventral striatum regulates motivation and reward (Grueter et al., 2012; Nestler 

et al., 2002; Russo et al., 2010). While the graft location findings from my thesis are not 

quantitative and need to be studied further, other evidence from clinical grafting trials 

provide insights into the possibility of graft location influencing GID induction. Therefore, 

a small preliminary animal experiment could be designed in which eVM neurons are 

grafted into specific regional locations of the striatum (e.g., dorsolateral vs. 

ventrolateral) and assessed for GID behavior. 

333 

 
 
Figure 5.3: Qualitative comparison of graft location and GID scores in each 
host/donor combination. 
Homozygous Met/Met parkinsonian rats engrafted with WT DA neurons (shown above in 
green) demonstrated a higher number of animals that exhibited increased GID behavior, 
and an increased number of these animals with GID behavior had grafts that were placed 
more dorsolateral in the striatum. The dotted line demarcates a total GID score of 15 or 
above at 10 weeks post-engraftment.  

Transplanting iPSCs into our rs6265 Parkinsonian Rat Model 

Previously discussed above, clinical trials are now shifting (see Table 5.2) toward 

utilizing iPSCs as a cell source for neural transplantation in PD due to the ethical 

concerns of using embryonic neurons and the obstacles of obtaining a sufficient amount 

of tissue (Barker et al., 2024). However, because VM transplants are currently the only 

cell source to induce GID behavior, the clinical outcomes of iPSC transplants remain 

unknown. Moreover, like past trials, iPSCs have not been genotyped for the rs6265 

SNP to the best of my knowledge, and therefore, it is unknown whether the host/donor 

interactions we have demonstrated in our preclinical parkinsonian rats also apply to 

iPSC transplants. Regardless, an advantage of using iPSCs is that they could be 

programmed and/or genetically manipulated to produce less, or more, DA and/or 

express less or more VMAT2/VGLUT2, if these are indeed the underlying factors 

responsible for GID behavior. It could, however, be entirely possible that transplantation 

of iPSCs do not induce GID behavior in DA-grafted parkinsonian individuals. Therefore, 

334 

 
 
I would recommend that a similar experiment to my studies be conducted in the future 

using iPSCs as the cell source for transplantation to determine whether these cells have 

the potential to induce GID.  

Table 5.2: Clinical Trials using iPSCs. 
Current planned or ongoing clinical trials using iPSCs as a cell source for neuron 
transplantation in PD. Abbreviations: iPSCs = induced pluripotent stem cells; PASCs = 
PASCs = pluripotent stem cells isolated from adipose tissue; DA = dopamine; HiPSC = 
human induced pluripotent stem cells; iPSC-DAPs = induced pluripotent stem cells 
dopaminergic progenitor cells. 

335 

Clinical Trial ID Location Cell Source Status NCT06687837 Boston, MA, USA Autologous iPSCs Recruiting NCT06482268 La Jolla, CA, USA Human iPSCs Recruiting NCT06422208 Boston, MA, USA Autologous iPSC-derived DA neurons Enrolling my invitation NCT06141317 San Jose, Costa Rica PASCs Active, not recruiting NCT05901818 Beijing, China Autologous induced neural stem cell-derived DA precursor cells Recruiting JMA-IIA00384 Kyoto, Japan Allogenic human iPSCs Completed NCT06145711 Shanghai, Shanghai China HiPSC-derived dopaminergic neural precursor cells Recruiting  NCT06821529 Hangzhou, Zhejiang, China iPSC-DAPs Not yet recruiting   
 
 
 
 
 
CONCLUDING REMARKS 

The key findings presented in this thesis are three-fold: (1) the homozygous 

rs6265 Met/Met genotype, regardless of host or donor, confers a degree of functional 

graft-derived benefit in parkinsonian rats; (2) homozygous rs6265 Met/Met parkinsonian 

rats engrafted with WT DA neurons remain the only host/donor combination to develop 

aberrant GID behavior; (3) excess DA release and/or DA/glutamate co-transmission are 

promising factors that likely underlie GID behavior. These findings ultimately support the 

notion that PD remains a complex, heterogeneous disorder, making it almost impossible 

to develop a “one-size-fits-all” therapy that works uniformly for everyone. Instead, a 

precision-medicine-based approach, especially in regenerative cell therapy, is 

warranted to treat PD. Results obtained from this thesis have provided a solid 

foundation for future studies moving forward in this precision-medicine-based climate.  

It is, again, highly recommended that PD patients and donors be genotyped for 

the rs6265 SNP prior to receiving cell transplants. Additionally, now that the field have 

shifted toward implementing iPSCs as a new cell source in clinical trials, we can now 

determine whether iPSC-engrafted patients develop GID. In the event that GID do 

develop, we now have at least some insight into the underlying GID mechanisms based 

on my thesis findings. While cell transplantation does not, and will not, offer a “cure” for 

PD, it does offer a promising non-pharmacological alternative to the therapies that are 

currently prescribed. However, until cell transplantation is completely optimized by 

harnessing the benefits while preventing the side effects (e.g., GID), neural 

transplantation will not be considered a fully safe and effective therapeutic alternative to 

treat PD. 

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